Polymer conjugates of interferon beta-1a and uses

ABSTRACT

An interferon beta polypeptide comprising interferon-beta 1a coupled to a polymer containing a polyalkylene glycol moiety wherein the interferon-beta-1a and the polyalkylene glycol moiety are arranged such that the interferon-beta-1a has an enhanced activity relative to another therapeutic form of interferon beta (interferon-beta-1b) and exhibits no decrease in activity as compared to non-conjugated interferon-beta-1a. The conjugates of the invention are usefully employed in therapeutic as well as non-therapeutic, e.g., diagnostic, applications.

RELATED APPLICATIONS

This is a continuation of PCT/US99/24201, filed on Oct. 15, 1999 as acontinuations-in-part of prior U.S. Provisional Ser. No. 60/104,572filed Oct. 16, 1998 and U.S. Provisional Ser. No. 60/120,161 filed Feb.16, 1999. The teachings of the earlier-filed patent applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Use of polypeptides and proteins for the systemic treatment of specificdiseases is now well accepted in medical practice. The role that thesesubstances play in therapy is so important that many research activitiesare being directed towards the synthesis of large quantities byrecombinant DNA technology. Many of these polypeptides are endogenousmolecules which are very potent and specific in eliciting theirbiological actions.

A major factor limiting the usefulness of these proteinaceous substancesfor their intended application is that, when given parenterally, theyare eliminated from the body within a short time. This can occur as aresult of metabolism by proteases or by clearance using normal pathwaysfor protein elimination such as by filtration in the kidneys. The oralroute of administration of these substances is even more problematicbecause in addition to proteolysis in the stomach, the high acidity ofthe stomach destroys them before they reach their intended targettissue. The problems associated with these routes of administration ofproteins are well known in the pharmaceutical industry, and variousstrategies are being used in attempts to solve them.

A great deal of work dealing with protein stabilization has beenpublished. Various ways of conjugating proteins with polymeric materialsare known, including use of dextrans, polyvinyl pyrrolidones,glycopeptides, polyethylene glycol and polyamino acids. The resultingconjugated polypeptides are reported to retain their biologicalactivities and solubility in water for parenteral applications.

A peptide family which has been the focus of much clinical work, andefforts to improve its administration and bio-assimilation, is theinterferons. Interferons have been tested in a variety of clinicaldisease states. The use of human interferon beta, one member of thatfamily, is best established in the treatment of multiple sclerosis. Twoforms of recombinant interferon beta, have recently been licensed inEurope and the U.S. for treatment of this disease. One form isinterferon-beta-1a (trademarked and sold as AVONEX®, mfg. Biogen, Inc.,Cambridge, Mass.) and hereinafter, “interferon-beta-1a” or “IFN-beta-1a”or “IFN-β-1a” or “interferon-β-1a”, used interchangeably. The other formis interferon-beta-1b (trademarked and sold as BETASERON®. Berlex,Richmond, Calif.), hereinafter, “interferon-beta-1b”. Interferon beta-1ais produced in mammalian cells using the natural human gene sequence andis glycosylated, whereas interferon beta-1b is produced in E. colibacteria using a modified human gene sequence that contains agenetically engineered cysteine-to-serine substitution at amino acidposition 17 and is non-glycosylated.

Previously, several of us have directly compared the relative in vitropotencies of interferon-beta-1a and interferon beta 1b in functionalassays and showed that the specific activity of interferon-beta-1a isapproximately 10-fold greater than the specific activity ofinterferon-beta-1b (Runkel et al., 1998, Pharm. Res. 15: 641-649). Fromstudies designed to identify the structural basis for these activitydifferences, we identified glycosylation as the only one of the knownstructural differences between the products that affected the specificactivity. The effect of the carbohydrate was largely manifested throughits stabilizing role on structure. The stabilizing effect of thecarbohydrate was evident in thermal denaturation experiments and SECanalysis. Lack of glycosylation was also correlated with an increase inaggregation and an increased sensitivity to thermal denaturation.Enzymatic removal of the carbohydrate from interferon-beta-1a withPNGase F caused extensive precipitation of the deglycosylated product.

These studies indicate that, despite the conservation in sequencebetween interferon-beta-1a and interferon-beta-1b, they are distinctbiochemical entities and therefore much of what is known aboutinterferon-beta-1b cannot be applied to interferon-beta-1a, and viceversa.

SUMMARY OF THE INVENTION

We have exploited the advantages of glycosylated interferon-betarelative to non-glycosylated forms. In particular, we have developed aninterferon-beta-1a composition with increased activity relative tointerferon-beta-1b and that also has the salutory properties ofpegylated proteins in general with no effective loss in activity ascompared to interferon-beta-1a forms that are not conjugated. Thus, ifmodifications are made in such a way that the products(polymer-interferon-beta 1a conjugates) retain all or most of theirbiological activities, the following properties may result: alteredpharmacokinetics and pharmacodynamics leading to increased half-life andalterations in tissue distribution (e.g, ability to stay in thevasculature for longer periods of time), increased stability insolution, reduced immunogenicity, protection from proteolytic digestionand subsequent abolition of activity. Such a formulation is asubstantial advance in the pharmaceutical and medical arts and wouldmake a significant contribution to the management of various diseases inwhich interferon has some utility, such as multiple sclerosis, fibrosis,and other inflammatory or autoimmune diseases, cancers, hepatitis andother viral diseases. In particular, the ability to remain for longerperiods of time in the vasculature allows the interferon beta 1a to beused to inhibit angiogenesis and potentially to cross the blood-brainbarrier. Further, the thermal stability gained by creatingpolymer-interferon-beta-1a conjugates is an advantage when formulatinginterferon-beta-1a in powder form for use in subsequent administrationvia inhalation.

We used our knowledge of the crystallographic structure ofinterferon-beta-1a and developed an interferon-beta-1a-polymer conjugatein which the polymer is linked to those interferon-beta-1a site(s) thatwill allow the conjugate to retain full activity of theinterferon-beta-1a as compared to interferon-beta-1a that is notconjugated.

One aspect of the invention is a conjugated interferon-beta-1a complexwherein the interferon-beta-1a is covalently bonded to a polymerincorporating as an integral part thereof a polyalkylene glycol.

In one particular aspect, the present invention relates to aphysiologically active interferon-beta-1a composition comprisingphysiologically active interferon-beta-1a coupled with a polymercomprising a polyalkylene glycol moiety wherein the interferon-beta-1aand polyalkylene glycol moiety are arranged such that thephysiologically active interferon-beta-1a in the composition has anenhanced half life relative to the interferon-beta-1a alone (i.e., in anunconjugated form devoid of the polymer coupled thereto).

Another aspect of the invention is an interferon-beta-1a compositioncomprising physiologically active interferon-beta-1a coupled with apolymer in which the interferon-beta-1a is a fusion protein, preferablyan immunoglobulin fusion. In such a complex, the close proximity of theN-terminus (site of conjugation with polymer) and the C-terminus (siteof fusion with the Ig moiety) suggests that polymer conjugation mayreduce the immunogenicity of the fusion protein.

In another aspect, the present invention relates to a physiologicallyactive interferon-beta-1a composition comprising physiologically activeinterferon-beta-1a coupled with a polymer comprising a polyalkyleneglycol moiety wherein the interferon-beta-1a and polyalkylene glycolmoiety are arranged such that the physiologically activeinterferon-beta-1a in the composition has an enhanced activity relativeto interferon-beta-1b alone (i.e., in an unconjugated form devoid of thepolymer coupled thereto).

Another embodiment of the invention is a conjugated interferon-beta-1aprotein whose interferon-beta-1a moiety has been mutated to provide formuteins with selectively enhanced antiviral and/or antiproliferativeactivity relative to non-mutated forms of interferon-beta-1a.

The invention relates to a further aspect to a stable, aqueouslysoluble, conjugated interferon-beta-1a complex comprising aphysiologically active interferon-beta-1a covalently coupled to aphysiologically compatible polyethylene glycol moiety. In such complex,the interferon-beta-1a may be covalently coupled to the physiologicallycompatible polyethylene glycol moiety by a labile covalent bond at afree amino acid group of the interferon-beta-1a, wherein the labilecovalent bond is severed in vivo by biochemical hydrolysis and/orproteolysis.

In another aspect, the present invention relates to a dosage formcomprising a pharmaceutically acceptable carrier and a stable, aqueouslysoluble, interferon-beta 1a complex comprising interferon-beta coupledto a physiologically compatible polyethylene glycol.

In another aspect, covalently coupled interferon-beta-1a compositionssuch as those described above may utilize interferon-beta-1a intendedfor diagnostic or in vitro applications, wherein the interferon-beta-1ais for example a diagnostic reagent for immunoassay or other diagnosticor non-in vivo applications. In such non-therapeutic applications, thecomplexes of the invention are highly usefully employed as stabilizedcompositions which may for example be formulated in compatible solventsor other solution-based formulations to provide stable compositionalforms which are of enhanced resistance to degradation.

Modification of interferon-beta 1a with a non-toxic polymer may offercertain advantages. If modifications are made in such a way that theproducts (polymer-interferon-beta 1a conjugates) retain all or most oftheir biological activities the following properties may result: alteredpharmacokinetics and pharmacodynamics leading to increased half-life andalterations in tissue distribution (e.g, ability to stay in thevasculature for longer periods of time), increased stability insolution, reduced immunogenicity, protection of the modifiedinterferon-beta 1a from proteolytic digestion and subsequent abolitionof activity; increased thermal stability leading to more effectiveformulation of powdered interferon-beta-1a for oral or inhaled use.

Interferon-beta-1a endowed with the improved properties described abovemay be effective as therapy following either oral, aerosol, orparenteral administration. Other routes of administration, such as nasaland transdermal, may also be possible using the modified interferon-beta1a.

Another aspect of the invention is a method of inhibiting angiogenesisand neovascularization comprising subject an effective amount of thecompositions of the invention. As a result of increasing the level andduration of the interferon in the vasculature, the pegylated product ofthe invention should be particularly effective as an angiogenesisinhibitor.

In non-therapeutic (e.g., diagnostic) applications, conjugation ofdiagnostic and/or reagent species of interferon-beta is alsocontemplated. The resulting conjugated agent is resistant toenvironmental degradative factors, including solvent- orsolution-mediated degradation processes. As a result of such enhancedresistance and increased stability of interferon-beta-1a, the stabilityof the active ingredient is able to be significantly increased, withconcomitant reliability of the interferon-beta-1a containing compositionin the specific end use for which same is employed.

Other aspects, features, and modifications of the invention will be morefully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Binding of alanine substituted interferon-beta-1a mutants to adimeric fusion protein comprising the extracellular domain of the type Iinterferon receptor chain, IFNAR2/Ig (IFNAR2 ectodomain fused to thehuman IgG1 constant domain.

The binding affinities of the alanine substituted IFN mutants (A1-E) forthe IFNAR2 receptor chain were determined as described in Example 1(subsection D). The histogram presents their binding affinities in thisassay relative to wild type his-IFN-beta (% w.t.). The % w.t. valueswere calculated as the (affinity of wild type his-IFN-beta)/affinity ofmutant IFN-beta×100. The % w.t. (O) for individual experiments (n=3) andan average % w.t. (x) for the experimental set are shown. Mutants A2,AB1, AB2, and E did not bind IFNAR2/Fc at concentrations 500-fold higherthan the w.t. his-IFN-beta EC 50 (*)

FIG. 2. Binding of alanine substituted interferon-beta-1a mutants to thetype I interferon cell surface receptor complexes (“IFNAR½ complex”)expressed on Daudi Burkitt's lymphoma cells. The receptor bindingproperties of the alanine substitution mutants (A1-E) were determinedusing a FACS based, cell surface receptor binding assay as described inExample 1(subsection D). The histogram presents their receptor bindingaffinities in this assay relative to wild type his-IFN-beta (% w.t.).The % w.t. for each mutant was calculated as the (affinity of wild typehis-IFN-beta)/affinity of mutant IFN-beta×100. The % w.t. values (O) forindividual experiments and an average of the % w.t. values for theexperimental set (x) are shown.

FIG. 3. Antiviral activities of alanine substituted interferon-beta-1amutants

The antiviral activities of the alanine substitution mutants (A1-E) weredetermined on human A549 cells challenged with EMC virus as described inExample 1 (subsection E). The histogram presents their activities inthis assay relative to wild type his-IFN-beta (% w.t.). The % w.t. wascalculated as the (concentration of w.t. his-IFN-beta [50%cpe]/concentration of mutant IFN-beta [50% cpe]×100. The % w.t ( ) formultiple assays and the average of the experimental data set (x) areshown.

FIG. 4. Antiproliferative activities of alanine substitutedinterferon-beta-1a mutants The antiproliferation activity of the alaninesubstitution mutants (A1-E) were determined on Daudi Birkitt's lymphomacells as described in Example 1 (subsection E). The histogram presentstheir activities in this assay relative to wild type his-IFN-beta (%w.t). The % w.t. was calculated as (concentration w.t his-IFN-beta [50%growth inhibition]/concentration of mutant IFN-beta [50% growthinhibition]×100. The % w.t (0) for multiple assays and the average ofthe experimental data set (x) are shown

FIG. 5. Relative antiviral and antiproliferative activities of alaninesubstituted interferon-beta-1a mutants. The relative activities ofalanine substitution mutants (A1-E) in the antiviral (x axis) andantiproliferation (y axis) assays were compared. The average percentwild type his-IFN-beta (% w.t., x) presented in FIGS. 3 and 4 were usedfor this comparison. Those mutants that display a coordinate change inboth activities would fall on the vertical line. Those mutants thatdisplay a change in antiviral activity that is disproportionate to thechange in antiproliferation activity fall significantly off the diagonalline (DE1, D, C1). Significance was determined from consideration ofstandard deviations inherent in the average % w.t. values used.

FIG. 6. Localization of the site of pegylation by peptide mapping.Pegylated and unmodified interferon-β-1a were subjected to peptidemapping analysis. Samples were digested with endoproteinase Lys-C andsubjected to reverse phase HPLC on a C₄ column. The column was developedwith a 0-70% gradient of acetonitrile in 0.1% trifluoroacetic acid. Thecolumn effluent was monitored at 214 nm. Panel a, unmodifiedinterferon-β-1a. Panel b, pegylated interferon-β-1a. Arrowheads mark theelution position of the N-terminal endoproteinase Lys peptide ofinterferon-β-1a containing amino acid resides 1-19.

FIG. 7. Antiviral Activity of Conjugated and Non-ConjugagedInterferon-beta-1a.

The activity of interferon-beta-1a or PEGylated interferon-beta-1a atthe concentrations indicated on the X axis were assessed in antiviralassays using human lung carcinoma (A549) cells challenged withencephalomyocarditis virus. Following a two day incubation with virus,viable cells were stained with MTT, the plates were read at 450 nm, andthe absorbance which is reflective of cell viability is shown on the Yaxis. The standard deviations are shown as error bars. The concentrationof interferon-beta-1a or PEGylated interferon beta-1a which offered 50%viral killing (the “50% cytopathic effect”) (50% maximum OD450) wasabout 11 pg/ml and the 50% cytopathic effect for PEGylatedinterferon-beta-1a was about 11 pg/ml.

FIG. 8. Assessing stabilization of conjugates using thermal denaturation

PEGylated interferon-beta-1a and untreated interferon-beta-1a control in20 mM HEPES pH 7.5, 20 mM NaCl were heated at a fixed rates of 1degree/min. Denaturation was followed by monitoring absorbance changesat 280 nm. (a) unmodified interferon-beta-1a (b) PEGylatedinterferon-beta-1a.

FIG. 9. Measurements of interferon-beta antiviral activity in the plasmaof mice treated with interferon-beta-1a or PEGylated interferon-beta-1a.

Mice are injected iv with either 50,000 Units of interferon-beta-1a or50,000 Units of pegylated-interferon-beta-1a (containing the 20K PEG).Blood from these mice is obtained via retro-orbital bleeds at varioustimes after interferon injection as indicated on the X axis. There areat least 3 mice bled at each time point, and plasma is prepared andfrozen until the time interferon-beta activity is evaluated in antiviralassays using human lung carcinoma (A549) cells challenged withencephalomyocarditis virus. Viable cells were stained with a solution ofMTT, the plates were read at 450 nm, to determine the absorbance whichis reflective of cell viability and interferon-beta activity. Standardcurves were generated for each plate using interferon-beta-1a and usedto determine the amount of interferon-beta activity in each sample. Datafrom the individual animals are shown.

FIG. 10. Full DNA sequence of histidine-tagged interferon beta gene andits protein product. The full DNA (SEQ ID NO: 1) and protein (SEQ ID NO:2) sequences of the histidine-tagged IFN-beta-1a are shown. The cleavedVCAM-1 signal sequence leaves 3 amino terminal residues (SerGlyGly)upstream of the histidine tag (His₆, positions 4-9). The enterokinaselinker sequence (AspAspAspAspLys) is separate from the histidine tag bya spacer (positions 10-12, SerSerGly). The natural IFN-beta-1a proteinsequence spans positions (Met18-Asn183).

FIG. 11. Schematic representation of overall cloning and expressionstrategy.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term “covalently coupled” means that the specifiedmoieties are either directly covalently bonded to one another, or elseare indirectly covalently joined to one another through an interveningmoiety or moieties, such as a bridge, spacer, or linkage moiety ormoieties.

Interferon—An “interferon” (also referred to as “IFN”) is a small,species-specific, single chain polypeptide, produced by mammalian cellsin response to exposure to a variety of inducers such as viruses,polypeptides, mitogens and the like. The most preferred interferon usedin the invention is glycosylated, human, interferon-beta that isglycosylated at residue 80 (Asn 80) and that is preferably derived viarecombinant DNA technologies. This preferred glycosylatedinterferon-beta is called “interferon-beta-1a” or “IFN-beta-1a” or“IFN-∃-1a” or “interferon beta 1a” or “interferon-∃-1a”, all usedinterchangeably. The term “interferon-beta-1a” is also meant toencompass mutants thereof (e.g., Example 1), provided that such mutantsare also glycosylated at residue 80 (Asn 80). Recombinant DNA methodsfor producing proteins, including interferons are known. See forexample, U.S. Pat. Nos. 4,399,216, 5,149,636, 5,179,017 (Axel et al) andU.S. Pat. No. 4,470,461 (Kaufman).

Preferred interferon-beta-1a polynucleotides that may be used in thepresent methods of the invention are derived from the wild-typeinterferon beta gene sequences of various vertebrates, preferablymammals and are obtained using methods that are well-known to thosehaving ordinary skill in the art such as the methods described in thefollowing U.S. Patents: U.S. Pat. No. 5,641,656 (issued Jun. 24, 1997:DNA encoding avian type I interferon proprotein and mature avian type Iinterferon), U.S. Pat. No. 5,605,688 (Feb. 25, 1997—recombinant dog andhorse type I interferons); U.S. Pat. No. 5,231,176 (Jul. 27, 1993, DNAmolecule encoding a human leukocyte interferon); U.S. Pat. No. 5,071,761(Dec. 10, 1991, DNA sequence coding for sub-sequences of humanlymphoblastoid interferons LyIFN-alpha-2 and LyIFN-alpha-3); U.S. Pat.No. 4,970,161 (Nov. 13, 1990, DNA sequence coding for humaninterferon-gamma); U.S. Pat. No. 4,738,931 (Apr. 19, 1988, DNAcontaining a human interferon beta gene); U.S. Pat. No. 4,695,543 (Sep.22, 1987, human alpha-interferon Gx-1 gene and U.S. Pat. No. 4,456,748(Jun. 26, 1984, DNA encoding sub-sequences of different, naturally,occurring leukocyte interferons).

Mutants of interferon-beta-1a may be used in accordance with thisinvention. Mutations are developed using conventional methods ofdirected mutagenesis, known to those of ordinary skill in the art.Moreover, the invention provides for functionally equivalentinterferon-beta-1a polynucleotides that encode for functionallyequivalent interferon-beta-1a polypeptides.

A first polynucleotide encoding interferon-beta-1a is “functionallyequivalent” compared with a second polynucleotide encodinginterferon-beta-1a if it satisfies at least one of the followingconditions:

(a): the “functional equivalent” is a first polynucleotide thathybridizes to the second polynucleotide under standard hybridizationconditions and/or is degenerate to the first polynucleotide sequence.Most preferably, it encodes a mutant interferon having the activity ofan interferon-beta-1a;

(b) the “functional equivalent” is a first polynucleotide that codes onexpression for an amino acid sequence encoded by the secondpolynucleotide.

In summary, the term “interferon” includes, but is not limited to, theagents listed above as well as their functional equivalents. As usedherein, the term “functional equivalent” therefore refers to aninterferon-beta-1a protein or a polynucleotide encoding theinterferon-beta-1a protein that has the same or an improved beneficialeffect on the mammalian recipient as the interferon of which it isdeemed a functional equivalent. As will be appreciated by one ofordinary skill in the art, a functionally equivalent protein can beproduced by recombinant techniques, e.g., by expressing a “functionallyequivalent DNA”. Accordingly, the instant invention embracesinterferon-beta-1a proteins encoded by naturally-occurring DNAs, as wellas by non-naturally-occurring DNAs which encode the same protein asencoded by the naturally-occurring DNA. Due to the degeneracy of thenucleotide coding sequences, other polynucleotides may be used to encodeinterferon-beta-1a. These include all, or portions of the abovesequences which are altered by the substitution of different codons thatencode the same amino acid residue within the sequence, thus producing asilent change. Such altered sequences are regarded as equivalents ofthese sequences. For example, Phe (F) is coded for by two codons, TTC orTTT, Tyr (Y) is coded for by TAC or TAT and His (H) is coded for by CACor CAT. On the other hand, Trp (W) is coded for by a single codon, TGG.Accordingly, it will be appreciated that for a given DNA sequenceencoding a particular interferon there will be many DNA degeneratesequences that will code for it. These degenerate DNA sequences areconsidered within the scope of this invention.

“fusion”—refers to a co-linear linkage of two or more proteins orfragments thereof via their individual peptide backbones through geneticexpression of a polynucleotide molecule encoding those proteins. It ispreferred that the proteins or fragments thereof be from differentsources. Thus, preferred fusion proteins include an interferon-beta-1aprotein or fragment covalently linked to a second moiety that is not aninterferon. Specifically, an “interferon-beta-1a/Ig fusion” is a proteincomprising an interferon-beta-1a molecule of the invention, or fragmentthereof linked to an N-terminus of an immunoglobulin chain wherein aportion of the N-terminus of the immunoglobulin is replaced with theinterferon-beta-1a.

“Recombinant,” as used herein, means that a protein is derived fromrecombinant, mammalian expression systems. Protein expressed in mostbacterial cultures, e.g., E. coli, will be free of glycan so theseexpression systems are not preferred. Protein expressed in yeast mayhave a oligosaccharide structures that are different from that expressedin mammalian cells.

“Biologically active,” as used throughout the specification as acharacteristic of interferon-beta 1a, means that a particular moleculeshares sufficient amino acid sequence homology with the embodiments ofthe present invention disclosed herein to be capable of antiviralactivity as measured in an in vitro antiviral assay of the type shown inExample 1 (see below).

A “therapeutic composition” as used herein is defined as comprising theproteins of the invention and other physiologically compatibleingredients. The therapeutic composition may contain excipients such aswater, minerals and carriers such as protein.

An “effective amount” of an agent of the invention is that amount whichproduces a result or exerts an influence on the particular conditionbeing treated.

“amino acid”—a monomeric unit of a peptide, polypeptide, or protein.There are twenty amino acids found in naturally occurring peptides,polypeptides and proteins, all of which are L-isomers. The term alsoincludes analogs of the amino acids and D-isomers of the protein aminoacids and their analogs.

A “derivatized” amino acid is a natural or nonnatural amino acid inwhich the normally occurring side chain or end group is modified bychemical reaction. Such modifications include, for example,gamma-carboxylation, beta-carboxylation, sulfation, sulfonation,phosphorylation, amidization, esterification, N-acetylation,carbobenzylation, tosylation, and other modifications known in the art.A “derivatized polypeptide” is a polypeptide containing one or morederivatized amino acids.

“protein”—any polymer consisting essentially of any of the 20 aminoacids. Although “polypeptide” is often used in reference to relativelylarge polypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and is varied.The term “protein” as used herein refers to peptides, proteins andpolypeptides, unless otherwise noted.

“mutant”—any change in the genetic material of an organism, inparticular any change (i.e., deletion, substitution, addition, oralteration) in a wild-type polynucleotide sequence or any change in awild-type protein. The term “mutein” is used interchangeably with“mutant”.

“wild-type”—the naturally-occurring polynucleotide sequence of an exonof a protein, or a portion thereof, or protein sequence, or portionthereof, respectively, as it normally exists in vivo.

“standard hybridization conditions”—salt and temperature conditionssubstantially equivalent to 0.5×SSC to about 5×SSC and 65° C. for bothhybridization and wash. The term “standard hybridization conditions” asused herein is therefore an operational definition and encompasses arange of hybridization conditions. Higher stringency conditions may, forexample, include hybridizing with plaque screen buffer (0.2%polyvinylpyrrolidone, 0.2% Ficoll 400; 0.2% bovine serum albumin, 50 mMTris-HCl (pH 7.5); 1 M NaCl; 0.1% sodium pyrophosphate; 1% SDS); 10%dextran sulfate, and 100 μg/ml denatured, sonicated salmon sperm DNA at65° C. for 12-20 hours, and washing with 75 mM NaCl/7.5 mM sodiumcitrate (0.5×SSC)/1% SDS at 65° C. Lower stringency conditions may, forexample, include hybridizing with plaque screen buffer, 10% dextransulfate and 110 μg/ml denatured, sonicated salmon sperm DNA at 55° C.for 12-20 hours, and washing with 300 mM NaCl/30 mM sodium citrate(2.0×SSC)/1% SDS at 55° C. See also Current Protocols in MolecularBiology, John Wiley & Sons, Inc. New York, Sections 6.3.1-6.3.6, (1989).

“expression control sequence”—a sequence of polynucleotides thatcontrols and regulates expression of genes when operatively linked tothose genes.

“operatively linked”—a polynucleotide sequence (DNA, RNA) is operativelylinked to an expression control sequence when the expression controlsequence controls and regulates the transcription and translation ofthat polynucleotide sequence. The term “operatively linked” includeshaving an appropriate start signal (e.g., ATG) in front of thepolynucleotide sequence to be expressed and maintaining the correctreading frame to permit expression of the polynucleotide sequence underthe control of the expression control sequence and production of thedesired polypeptide encoded by the polynucleotide sequence.

“expression vector”—a polynucleotide, such as a DNA plasmid or phage(among other common examples) which allows expression of at least onegene when the expression vector is introduced into a host cell. Thevector may, or may not, be able to replicate in a cell.

“Isolated” (used interchangeably with “substantially pure”)—when appliedto nucleic acid i.e., polynucleotide sequences, that encodepolypeptides, means an RNA or DNA polynucleotide, portion of genomicpolynucleotide, cDNA or synthetic polynucleotide which, by virtue of itsorigin or manipulation: (i) is not associated with all of apolynucleotide with which it is associated in nature (e.g., is presentin a host cell as an expression vector, or a portion thereof); or (ii)is linked to a nucleic acid or other chemical moiety other than that towhich it is linked in nature; or (iii) does not occur in nature. By“isolated” it is further meant a polynucleotide sequence that is: (i)amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) chemically synthesized; (iii) recombinantly produced by cloning; or(iv) purified, as by cleavage and gel separation.

Thus, “substantially pure nucleic acid” is a nucleic acid which is notimmediately contiguous with one or both of the coding sequences withwhich it is normally contiguous in the naturally occurring genome of theorganism from which the nucleic acid is derived. Substantially pure DNAalso includes a recombinant DNA which is part of a hybrid gene encodingadditional sequences.

“Isolated” (used interchangeably with “substantially pure”)—when appliedto polypeptides means a polypeptide or a portion thereof which, byvirtue of its origin or manipulation: (i) is present in a host cell asthe expression product of a portion of an expression vector; or (ii) islinked to a protein or other chemical moiety other than that to which itis linked in nature; or (iii) does not occur in nature. By “isolated” itis further meant a protein that is: (i) chemically synthesized; or (ii)expressed in a host cell and purified away from associated proteins. Theterm generally means a polypeptide that has been separated from otherproteins and nucleic acids with which it naturally occurs. Preferably,the polypeptide is also separated from substances such as antibodies orgel matrices (polyacrylamide) which are used to purify it.

“heterologous promoter”—as used herein is a promoter which is notnaturally associated with a gene or a purified nucleic acid.

“Homologous”—as used herein is synonymous with the term “identity” andrefers to the sequence similarity between two polypeptides, molecules orbetween two nucleic acids. When a position in both of the two comparedsequences is occupied by the same base or amino acid monomer subunit(for instance, if a position in each of the two DNA molecules isoccupied by adenine, or a position in each of two polypeptides isoccupied by a lysine), then the respective molecules are homologous atthat position. The percentage homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared×100. Forinstance, if 6 of 10 of the positions in two sequences are matched orare homologous, then the two sequences are 60% homologous. By way ofexample, the DNA sequences CTGACT and CAGGTT share 50% homology (3 ofthe 6 total positions are matched). Generally, a comparison is made whentwo sequences are aligned to give maximum homology. Such alignment canbe provided using, for instance, the method of Needleman et al., J. MolBiol. 48: 443-453 (1970), implemented conveniently by computer programssuch as the Align program (DNAstar, Inc.). Homologous sequences shareidentical or similar amino acid residues, where similar residues areconservative substitutions for, or “allowed point mutations” of,corresponding amino acid residues in an aligned reference sequence. Inthis regard, a “conservative substitution” of a residue in a referencesequence are those substitutions that are physically or functionallysimilar to the corresponding reference residues, e.g., that have asimilar size, shape, electric charge, chemical properties, including theability to form covalent or hydrogen bonds, or the like. Particularlypreferred conservative substitutions are those fulfilling the criteriadefined for an “accepted point mutation” in Dayhoff et al., 5: Atlas ofProtein Sequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat.Biomed. Res. Foundation, Washington, D.C. (1978).

The terms polynucleotide sequence” and “nucleotide sequence” are alsoused interchangeably herein.

“angiogenesis” and “neovascularization” means, in their broadest sense,the recruitment of new blood vessels. In particular, angiogenesis alsorefers to the recruitment of new blood vessels at a tumor site.

“IFNAR2”, “IFNAR1”, “IFNAR½” refer to the proteins knows to compose thecell surface type I interferon receptor. The extracellular portion(ectodomain) portion of the IFNAR2 chain alone can bind interferon alphaor beta.

Practice of the present invention will employ, unless indicatedotherwise, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, protein chemistry, andimmunology, which are within the skill of the art. Such techniques aredescribed in the literature. See, for example, Molecular Cloning: ALaboratory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.),Cold Spring Harbor Laboratory Press, 1989; DNA Cloning, Volumes I and II(D. N. Glover, ed), 1985; Oligonucleotide Synthesis, (M. J. Gait, ed.),1984; U.S. Pat. No. 4,683,195 (Mullis et al.,); Nucleic AcidHybridization (B. D. Hames and S. J. Higgins, eds.), 1984; Transcriptionand Translation (B. D. Hames and S. J. Higgins, eds.), 1984; Culture ofAnimal Cells (R. I. Freshney, ed). Alan R. Liss, Inc., 1987; ImmobilizedCells and Enzymes, IRL Press, 1986; A Practical Guide to MolecularCloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and 155(Wu et al., eds), Academic Press, New York; Gene Transfer Vectors forMammalian Cells (J. H. Miller and M. P. Calos, eds.), 1987, Cold SpringHarbor Laboratory; Immunochemical Methods in Cell and Molecular Biology(Mayer and Walker, eds.), Academic Press, London, 1987; Handbook ofExperiment Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds.), 1986; Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, 1986.

The Interferon-beta

Interferon-beta-1a is useful as an agent for the treatment, remission orattenuation of a disease state, physiological condition, symptoms, oretiological factors, or for the evaluation or diagnosis thereof. Theterm also refers to interferon-beta-1a that is itself part of a fusionprotein such as an immunoglobulin-interferon-beta-1a fusion protein, asdescribed in co-pending applications Ser. Nos. 60/104,572 and60/120,161. Preparation of fusion proteins generally are well within theknowledge of persons having ordinary skill in the art.

We found unique site(s) for polymer attachment that would not destroyfunction of the interferon-beta-1a. In addition, we also usedsite-directed mutagenesis methods to independently investigate site(s)for polymer attachment (See Example 1). Briefly, we undertook amutational analysis of human interferon-beta-1a with the aim of mappingresides required for activity and receptor binding. The availability ofthe 3-D crystal structure of human interferon-beta-1a (see above andExample 1) allows us to identify, for alanine (or serine) substitutions,the solvent-exposed residues available for interferon beta receptorinteractions, and to retain amino acids involved in intramolecularbonds. A panel of fifteen alanine scanning mutations were designed thatreplaced between two and eight residues along distinct regions of eachof the helices (A (A1 (SEQ ID NO:26), A2 (SEQ ID NO:27)), B (B1(SEQ IDNO:31), B2(SEQ ID NO:32), C (C1 (SEQ ID NO:33), C2 (SEQ ID NO:34)), D(SEQ ID NO:37), E (SEQ ID NO:40)) and loops (AB1(SEQ ID NO:28), AB2 (SEQID NO:29), AB3 (SEQ ID NO:30), CD1 (SEQ ID NO:35), CD2 (SEQ ID NO:36),DE1 (SEQ ID NO:38), DE2 (SEQ ID NO:39) of interferon-beta-1a (SEQ IDNO:25). See Example 1.

An amino-terminal histidine tag (“his” tag) was included for affinitypurification of mammalian cell expressed mutants (FIG. 10 and SEQ IDNOS: 1 and 2 for the cDNA and deduced amino acid sequences,respectively) Functional consequences of these mutations are assessed inantiviral and antiproliferation assays. A non-radioactive binding assaywas developed to analyze these mutants for their binding to theinterferon beta surface cell receptor (IFNAR½ cell surface receptor). Inaddition, an ELISA-based assay employing an IFNAR2-ectodomain/Ig fusionprotein to bind interferon was used to map interactions of surfacesbetween interferon-beta-1a and IFNAR2 (See Example 1). These mutationalanalyses demonstrated that N- and C-termini lie in a portion of theinterferon-beta molecule not important for receptor binding orbiological function.

The mutants are further variants of the interferon beta 1a moiety of theinvention that may be particularly useful inasmuch as they display novelproperties not found in the wild type interferon-beta-1a (See Example1). We have identified three types of effects that were caused bytargeted mutagenesis. These effects may be advantageous for interferondrug development under certain circumstances. The three types of effectare as follows: (a) mutants with higher antiviral activity that ofhis-wild-type interferon-beta-1a (e.g. mutant C1); (b) mutants whichdisplay activity in both antiviral and antiproliferation assays, but forwhich antiproliferation activity is disproportionately low with respectto antiviral activity, compared to his-wild-type interferon-beta-1a(e.g., mutants C1, D and DE1); and (c) functional antagonists (e.g., A1,B2, CD2 and DE1), which show antiviral and antiproliferative activitiesthat are disproportionately low with respect to receptor binding,compared to his-wild-type interferon-beta-1a.

The Polymer Moiety

Within the broad scope of the present invention, a single polymermolecule may be employed for conjugation with an interferon-beta 1a,although it is also contemplated that more than one polymer molecule canbe attached as well. Conjugated interferon-beta 1a compositions of theinvention may find utility in both in vivo as well as non-in vivoapplications. Additionally, it will be recognized that the conjugatingpolymer may utilize any other groups, moieties, or other conjugatedspecies, as appropriate to the end use application. By way of example,it may be useful in some applications to covalently bond to the polymera functional moiety imparting UV-degradation resistance, orantioxidation, or other properties or characteristics to the polymer. Asa further example, it may be advantageous in some applications tofunctionalize the polymer to render it reactive or cross-linkable incharacter, to enhance various properties or characterisics of theoverall conjugated material. Accordingly, the polymer may contain anyfunctionality, repeating groups, linkages, or other constitutentstructures which do not preclude the efficacy of the conjugatedinterferon-beta 1a composition for its intended purpose. Otherobjectives and advantages of the present invention will be more fullyapparent from the ensuing disclosure and appended claims.

Illustrative polymers that may usefully be employed to achieve thesedesirable characteristics are described herein below in exemplaryreaction schemes. In covalently bonded peptide applications, the polymermay be functionalized and then coupled to free amino acid(s) of thepeptide(s) to form labile bonds.

The interferon-beta-1a is conjugated most preferably via a terminalreactive group on the polymer although conjugations can also be branchedfrom the non-terminal reactive groups. The polymer with the reactivegroup(s) is designated herein as “activated polymer”. The reactive groupselectively reacts with free amino or other reactive groups on theprotein. The activated polymer(s) are reacted so that attachment mayoccur at any available interferon-beta-1a amino group such as the alphaamino groups or the epsilon-amino groups of lysines. Free carboxylicgroups, suitably activated carbonyl groups, hydroxyl, guanidyl, oxidizedcarbohydrate moieties and mercapto groups of the interferon-beta-1a (ifavailable) can also be used as attachment sites.

Although the polymer may be attached anywhere on the interferon-beta 1amolecule, the most preferred site for polymer coupling is the N-terminusof the interferon-beta-1a. Secondary site(s) are at or near theC-terminus and through sugar moieties. Thus, the invention contemplatesas its most preferred embodiments: (i) N-terminally coupled polymerconjugates of interferon-beta-1a; (ii) C-terminally coupled polymerconjugates of interferon-beta-1a; (iii) sugar-coupled conjugates ofpolymer conjugates; (iv) as well as N-, C- and sugar-coupled polymerconjugates of interferon-beta-1a fusion proteins.

Generally from about 1.0 to about 10 moles of activated polymer per moleof protein, depending on protein concentration, is employed. The finalamount is a balance between maximizing the extent of the reaction whileminimizing non-specific modifications of the product and, at the sametime, defining chemistries that will maintain optimum activity, while atthe same time optimizing, if possible, the half-life of the protein.Preferably, at least about 50% of the biological activity of the proteinis retained, and most preferably 100% is retained.

The reactions may take place by any suitable method used for reactingbiologically active materials with inert polymers, preferably at aboutpH 5-7 if the reactive groups are on the alpha amino group at theN-terminus. Generally the process involves preparing an activatedpolymer (that may have at least one terminal hydroxyl group) andthereafter reacting the protein with the activated polymer to producethe soluble protein suitable for formulation. The above modificationreaction can be performed by several methods, which may involve one ormore steps.

As mentioned above, the most preferred embodiments of the inventionutilize the N-terminal end of interferon-beta-1a as the linkage to thepolymer. Suitable methods are available to selectively obtain anN-terminally modified interferon-beta-1a. One method is exemplified by areductive alkylation method which exploits differential reactivity ofdifferent types of primary amino groups (the epsilon amino groups on thelysine versus the amino groups on the N-terminal methionine) availablefor derivatization on interferon-beta-1a. Under the appropriateselection conditions, substantially selective derivatization ofinterferon-beta-1a at its N-terminus with a carbonyl group containingpolymer can be achieved. The reaction is performed at a pH which allowsone to take advantage of the pKa differences between the epsilon-aminogroups of the lysine residues and that of the alpha-amino group of theN-terminal residue of interferon-beta-1a. This type of chemistry is wellknown to persons with ordinary skill in the art.

We used a reaction scheme in which this selectivity is maintained byperforming reactions at low pH (generally 5-6) under conditions where aPEG-aldehyde polymer is reacted with interferon-beta-1a in the presenceof sodium cyanoborohydride. This results, after purification of thePEG-interferon-beta-1a and analysis with SDS-PAGE, MALDI massspectrometry and peptide sequencing/mapping, resulted in aninterferon-beta-1a whose N-terminus is specifically targeted by the PEGmoiety.

The crystal structure of interferon-beta-1a us such that the N- andC-termini are located close to each other (see Karpusas et al., 1997,Proc. Natl. Acad. Sci. 94: 11813-11818). Thus, modifications of theC-terminal end of interferon-beta-1a should also have minimal effect onactivity. While there is no simple chemical strategy for targeting apolyalkylene glycol polymer such as PEG to the C-terminus, it would bestraightforward to genetically engineer a site that can be used totarget the polymer moiety. For example, incorporation of a Cys at a sitethat is at or near the C-terminus would allow specific modificationusing a maleimide, vinylsulfone or haloacetate—activated polyalkyleneglycol (e.g., PEG). These derivatives can be used specifically formodification of the engineered cysteines due to the high selectively ofthese reagents for Cys. Other strategies such as incorporation of ahistidine tag which can be targeted (Fancy et al., (1996) Chem. & Biol.3: 551) or an additional glycosylation site, represent otheralternatives for modifying the C-terminus of interferon-beta-1a.

The glycan on the interferon-beta-1a is also in a position that wouldallow further modification without altering activity. Methods fortargeting sugars as sites for chemical modification are also well knownand therefore it is likely that a polyalkylene glycol polymer can beadded directly and specifically to sugars on interferon-beta-1a thathave been activated through oxidation. For example, apolyethyleneglycol-hydrazide can be generated which forms relativelystable hydrazone linkages by condensation with aldehydes and ketones.This property has been used for modification of proteins throughoxidized oligosaccharide linkages. See Andresz, H. et al., (1978),Makromol. Chem. 179: 301. In particular, treatment of PEG-carboxymethylhydrazide with nitrite produces PEG-carboxymethyl azide which is anelectrophilically active group reactive toward amino groups. Thisreaction can be used to prepare polyalkylene glycol-modified proteins aswell. See, U.S. Pat. Nos. 4,101,380 and 4,179,337.

We had previously discovered that thiol linker-mediated chemistry couldfurther facilitate cross-linking of proteins. In particular, wegenerated homotypic multimers of LFA-3 and CD4 using a procedure such asgenerating reactive aldehydes on carbohydrate moieties with sodiumperiodate, forming cystamine conjugates through the aldehydes andinducing cross-linking via the thiol groups on the cystamines. SeePepinsky, B. et al., (1991), J. Biol. Chem., 266: 18244-18249 and Chen,L. L. et al., (1991) J. Biol. Chem., 266: 18237-18243. Therefore, weenvision that this type of chemistry would also be appropriate formodification with polyalkylene glycol polymers where a linker isincorporated into the sugar and the polyalkylene glycol polymer isattached to the linker. While aminothiol or hydrazine-containing linkerswill allow for addition of a single polymer group, the structure of thelinker can be varied so that multiple polymers are added and/or that thespatial orientation of the polymer with respect to theinterferon-beta-1a is changed.

In the practice of the present invention, polyalkylene glycol residuesof C1-C4 alkyl polyalkylene glycols, preferably polyethylene glycol(PEG), or poly(oxy)alkylene glycol residues of such glycols areadvantageously incorporated in the polymer systems of interest. Thus,the polymer to which the protein is attached can be a homopolymer ofpolyethylene glycol (PEG) or is a polyoxyethylated polyol, provided inall cases that the polymer is soluble in water at room temperature.Non-limiting examples of such polymers include polyalkylene oxidehomopolymers such as PEG or polypropylene glycols, polyoxyethylenatedglycols, copolymers thereof and block copolymers thereof, provided thatthe water solubility of the block copolymer is maintained. Examples ofpolyoxyethylated polyols include, for example, polyoxyethylatedglycerol, polyoxyethylated sorbitol, polyoxyethylated glucose, or thelike. The glycerol backbone of polyoxyethylated glycerol is the samebackbone occurring naturally in, for example, animals and humans inmono-, di-, and triglycerides. Therefore, this branching would notnecessarily be seen as a foreign agent in the body.

As an alternative to polyalkylene oxides, dextran, polyvinylpyrrolidones, polyacrylamides, polyvinyl alcohols, carbohydrate-basedpolymers and the like may be used. Those of ordinary skill in the artwill recognize that the foregoing list is merely illustrative and thatall polymer materials having the qualities described herein arecontemplated.

The polymer need not have any particular molecular weight, but it ispreferred that the molecular weight be between about 300 and 100,000,more preferably between 10,000 and 40,000. In particular, sizes of20,000 or more are best at preventing protein loss due to filtration inthe kidneys.

Polyalkylene glycol derivatization has a number of advantageousproperties in the formulation of polymer-interferon-beta 1a conjugatesin the practice of the present invention, as associated with thefollowing properties of polyalkylene glycol derivatives: improvement ofaqueous solubility, while at the same time eliciting no antigenic orimmunogenic response; high degrees of biocompatibility; absence of invivo biodegradation of the polyalkylene glycol derivatives; and ease ofexcretion by living organisms.

Moreover, in another aspect of the invention, one can utilizeinterferon-beta 1a covalently bonded to the polymer component in whichthe nature of the conjugation involves cleavable covalent chemicalbonds. This allows for control in terms of the time course over whichthe polymer may be cleaved from the interferon-beta 1a. This covalentbond between the interferon-beta-1a drug and the polymer may be cleavedby chemical or enzymatic reaction. The polymer-interferon-beta-1aproduct retains an acceptable amount of activity. Concurrently, portionsof polyethylene glycol are present in the conjugating polymer to endowthe polymer-interferon-beta-1a conjugate with high aqueous solubilityand prolonged blood circulation capability. As a result of theseimproved characteristics the invention contemplates parenteral, nasal,and oral delivery of both the active polymer-interferon-beta-1a speciesand, following hydrolytic cleavage, bioavailability of theinterferon-beta-1a per se, in in vivo applications.

It is to be understood that the reaction schemes described herein areprovided for the purposes of illustration only and are not to belimiting with respect to the reactions and structures which may beutilized in the modification of the interferon-beta-1a, e.g., to achievesolubility, stabilization, and cell membrane affinity for parenteral andoral administration. The reaction of the polymer with theinterferon-beta 1a to obtain the most preferred N-terminal conjugatedproducts is readily carried out using a wide variety of reactionschemes. The activity and stability of the interferon-beta-1a conjugatescan be varied in several ways, by using a polymer of different molecularsize. Solubilities of the conjugates can be varied by changing theproportion and size of the polyethylene glycol fragment incorporated inthe polymer composition.

Utilities

The unique property of polyalkylene glycol-derived polymers of value fortherapeutic applications of the present invention is their generalbiocompatibility. The polymers have various water solubility propertiesand are not toxic. They are believed non-immunogenic and non-antigenicand do not interfere with the biological activities of theinterferon-beta-1a moiety when conjugated under the conditions describedherein. They have long circulation in the blood and are easily excretedfrom living organisms.

The products of the present invention have been found useful insustaining the half life of therapeutic interferon-beta 1a, and may forexample be prepared for therapeutic administration by dissolving inwater or acceptable liquid medium. Administration is by either theparenteral, aerosol, or oral route. Fine colloidal suspensions may beprepared for parenteral administration to produce a depot effect, or bythe oral route while aerosol formulation may be liquid or dry powder innature. In the dry, lyophilized state or in solution formulations, theinterferon-beta-1a-polymer conjugates of the present invention shouldhave good storage stability. The thermal stability of conjugatedinterferon-beta-1a (Example 3) is advantageous in powder formulationprocesses that have a dehydration step. See, e.g., PCT/US/95/06008(“Methods and Compositions for Dry Powder of Interferons”).

The therapeutic polymer conjugates of the present invention may beutilized for the prophylaxis or treatment of any condition or diseasestate for which the interferon-beta-1a constituent is efficacious. Inaddition, the polymer-based conjugates of the present invention may beutilized in diagnosis of constituents, conditions, or disease states inbiological systems or specimens, as well as for diagnosis purposes innon-physiological systems.

In therapeutic usage, the present invention contemplates a method oftreating an animal subject having or latently susceptible to suchcondition(s) or disease state(s) and in need of such treatment,comprising administering to such animal an effective amount of a polymerconjugate of the present invention which is therapeutically effectivefor said condition or disease state. Subjects to be treated by thepolymer conjugates of the present invention include mammalian subjectsand most preferably human subjects. Depending on the specific conditionor disease state to be combated, animal subjects may be administeredpolymer conjugates of the invention at any suitable therapeuticallyeffective and safe dosage, as may readily be determined within the skillof the art, and without undue experimentation. Because of the speciesbarriers of Type I interferons, it may be necessary to generateinterferon-polymer conjugates as described herein with interferons fromthe appropriate species.

The anti-cell proliferative activity of interferon-beta-1a is wellknown. In particular, certain of the interferon-beta-1a polymerconjugates described herein are useful for treating tumors and cancerssuch as osteogenic sarcoma, lymphoma, acute lymphocytic leukemia, breastcarcinoma, melanoma and nasopharyngeal carcinoma, as well as autoimmuneconditions such as fibrosis, lupus and multiple sclerosis. It is furtherexpected that the anti-viral activity exhibited by the conjugatedproteins, in particular certain of the interferon-beta-1a muteinconjugates described herein, may be used in the treatment of viraldiseases, such as ECM infection, influenza, and other respiratory tractinfections, rabies, and hepatitis. It is also expected thatimmunomodulatory activities of interferon-beta-1a exhibited by theconjugated proteins described herein, may be used in the treatment ofautoimmune and inflammatory diseases, such as fibrosis, multiplesclerosis. The ability of interferons to inhibit formation of new bloodvessels (i.e., inhibit angiogenesis and neovascularization) enablesconjugates of the invention to be used to treat angiogenic diseases suchas diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis and Osler-Webber Syndrome.

Moreover, the antiendothelial activity of interferon has been known forsome time and one potential mechanism of interferon action may be tointerfere with endothelial cell activity by inhibiting the production orefficacy of angiogenic factors produced by tumor cells. Some vasculartumors, such as hemangiomas, are particularly sensitive to treatmentwith interferon. Treatment with interferon-alpha is the only documentedtreatment for this disease. It is expected that treatment with theinterferon-beta-1a conjugates of the invention will offer subtantialpharmaceutical benefits in terms of pharmacokinetics andpharmacodynamics, since the conjugate is expected to remain in thevasculature for a longer period of time than non-conjugated interferons,thus leading to more efficient and effective therapy for use as ananti-angiogenic agent. See Example 8.

The polymer-interferon-beta-1a conjugates of the invention may beadministered per se as well as in the form of pharmaceuticallyacceptable esters, salts, and other physiologically functionalderivatives thereof. In such pharmaceutical and medicament formulations,the interferon-beta-1a preferably is utilized together with one or morepharmaceutically acceptable carrier(s) and optionally any othertherapeutic ingredients. The carrier(s) must be pharmaceuticallyacceptable in the sense of being compatible with the other ingredientsof the formulation and not unduly deleterious to the recipient thereof.The interferon-beta-1a is provided in an amount effective to achieve thedesired pharmacological effect, as described above, and in a quantityappropriate to achieve the desired daily dose.

The formulations include those suitable for parenteral as well asnon-parenteral administration, and specific administration modalitiesinclude oral, rectal, buccal, topical, nasal, ophthalmic, subcutaneous,intramuscular, intravenous, transdermal, intrathecal, intra-articular,intra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, andintra-uterine administration. Formulations suitable for oral, nasal, andparenteral administration are preferred.

When the interferon-beta-1a is utilized in a formulation comprising aliquid solution, the formulation advantageously may be administeredorally or parenterally. When the interferon-beta-1a is employed in aliquid suspension formulation or as a powder in a biocompatible carrierformulation, the formulation may be advantageously administered orally,rectally, or bronchially.

When the interferon-beta-1a is utilized directly in the form of apowdered solid, the interferon-beta-1a may advantageously beadministered orally. Alternatively, it may be administered nasally orbronchially, via nebulization of the powder in a carrier gas, to form agaseous dispersion of the powder which is inspired by the patient from abreathing circuit comprising a suitable nebulizer device.

The formulations comprising the polymer conjugates of the presentinvention may conveniently be presented in unit dosage forms and may beprepared by any of the methods well known in the art of pharmacy. Suchmethods generally include the step of bringing the active ingredient(s)into association with a carrier which constitutes one or more accessoryingredients. Typically, the formulations are prepared by uniformly andintimately bringing the active ingredient(s) into association with aliquid carrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into dosage forms of the desiredformulation.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,or lozenges, each containing a predetermined amount of the activeingredient as a powder or granules; or a suspension in an aqueous liquoror a non-aqueous liquid, such as a syrup, an elixir, an emulsion, or adraught.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which optionally is mixedwith a binder, disintegrant, lubricant, inert diluent, surface activeagent, or discharging agent. Molded tablets comprised of a mixture ofthe powdered polymer conjugates with a suitable carrier may be made bymolding in a suitable machine.

A syrup may be made by adding the active compound to a concentratedaqueous solution of a sugar, for example sucrose, to which may also beadded any accessory ingredient(s). Such accessory ingredient(s) mayinclude flavorings, suitable preservative, agents to retardcrystallization of the sugar, and agents to increase the solubility ofany other ingredient, such as a polyhydroxy alcohol, for exampleglycerol or sorbitol.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active conjugate, whichpreferably is isotonic with the blood of the recipient (e.g.,physiological saline solution). Such formulations may include suspendingagents and thickening agents or other microparticulate systems which aredesigned to target the compound to blood components or one or moreorgans. The formulations may be presented in unit-dose or multi-doseform.

Nasal spray formulations comprise purified aqueous solutions of theactive conjugate with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucus membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, hydrogenated fats, orhydrogenated fatty carboxylic acid.

Ophthalmic formulations such as eye drops are prepared by a similarmethod to the nasal spray, except that the pH and isotonic factors arepreferably adjusted to match that of the eye.

Topical formulations comprise the conjugates of the invention dissolvedor suspended in one or more media, such as mineral oil, petroleum,polyhydroxy alcohols, or other bases used for topical pharmaceuticalformulations.

In addition to the aforementioned ingredients, the formulations of thisinvention may further include one or more accessory ingredient(s)selected from diluents, buffers, flavoring agents, disintegrants,surface active agents, thickeners, lubricants, preservatives (includingantioxidants), and the like.

Accordingly, the present invention contemplates the provision ofsuitable polymers for in vitro stabilization of interferon-beta 1a insolution, as a preferred illustrative application of non-therapeuticapplication. The polymers may be employed for example to increase thethermal stability and enzymic degradation resistance of theinterferon-beta 1a. Enhancement of the thermal stability characteristicof the interferon-beta-1a via conjugation in the manner of the presentinvention provides a means of improving shelf life, room temperaturestability, and robustness of research reagents and kits.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof. In particular, it willbe understood that the in vivo, animal experiments described herein maybe varied, so that other modifications and variations of the basicmethodology are possible. For example, in Example 5, one of ordinaryskill in the art could use other neopterin assays or could alter thenumber and kind of primate used. These modifications and variations tothe Examples are to be regarded as being within the spirit and scope ofthe invention.

EXAMPLE 1 Structure/activity Studies of Human Interferon-beta-1a UsingAlanine/serine Substitution Mutations Analysis of Receptor Binding Sitesand Functional Domains

A. Overview

An extensive mutational analysis of human interferon-beta-1a(IFN-beta-1a) was undertaken with the aims of mapping residues requiredfor activity and receptor binding. The availability of the 3-D crystalstructure of human IFN-beta (Karpusas, M. et al. 1997, Proc. Natl. Acad.Sci. 94: 11813-11818) allowed us to identify for alanine (or serine)substitutions the solvent-exposed residues available for receptorinteractions, and to retain amino acids involved in intramolecularbonds. A panel of 15 alanine substitution mutations were designed thatreplaced between 2 and 8 residues along distinct regions of each of thehelices (A, B, C, D, E) and loops (AB, CD, DE). An amino-terminalhistidine tag comprising six histidine residues was included foraffinity purification, as well as an enterokinase cleavage site forremoval of the amino-terminal extension. The resulting interferons arereferred to as “his tagged-interferon(IFN)-beta” or“His-interferon-beta” or “His₆-interferon-beta” and the like.

Various mutant his tagged-IFN-beta expression plasmids were constructedusing a wild type IFN-beta gene construct as a template for mutagenesis.The mutagenesis strategy involved first introducing unique restrictionenzyme cleavage sites throughout the wild type his tagged-IFN beta gene,then replacing distinct DNA sequences between the chosen restrictionsites with synthetic oligonucleotide duplexes, which encoded the alanine(or serine) substitution mutations. Finally, the mutant IFN genes weresubcloned into a plasmid which directed mammalian cell expression in ahuman 293 kidney cell line.

Functional consequences of these mutations were assessed in antiviraland antiproliferation assays. A non-radioactive IFN binding assay wasdeveloped to analyze these mutants in their binding to the surfacereceptor (“IFNAR½ complex”) of human Daudi Burkitt's lymphoma cells. Inaddition, an assay to map interaction surfaces between his-IFN-betamutants and IFNAR2 was developed that employed a IFNAR2/Ig fusionprotein, comprised of the IFN receptor protein IFNAR2 extracellulardomain fused to the hinge, CH2 and CH3 domains of human IgG1.

-   1. Creation of an interferon beta gene as a template for mutagenesis

Our strategy to generate IFN-beta alanine (or serine) substitutedmutants was to first create a modified IFN-beta gene, which encoded thewild type protein but which carried unique restriction enzyme cleavagesites scattered across the gene. The unique sites were used to exchangewild type sequences for synthetic oligonucleotide duplexes, which encodethe mutated codons. In order to obtain an human IFN-beta-1a expressioncassette suitable for creation of mutant genes, the IFN-beta cDNA(GenBank accession #E00029) was amplified by PCR. An initial cloning ofthe IFN-beta gene into plasmid pMJB107, a derivative of pACYC184, seeRose, et. al., 1988, Nucleic Acids Res. 16 (1) 355) was necessary inorder to perform site-directed mutagenesis of the gene in a plasmid thatlacked the specific restriction sites which would be generated throughthe mutagenesis.

The PCR primers used to subclone the coding sequences of the humanIFN-beta gene also allowed us to introduce an enterokinase cleavage siteupstream and in frame with the IFN-beta gene

(5′ PCR primer 5′TTCTCCGGAGACGATGATGACAAGATGAGCTACAACTT (SEQ ID NO: 3:“BET-021”, and GCTTGGATTCCTACAAAGAAGC-3′ 3′ PCR primer5′-GCCGCTCGAGTTATCAGTTTCGGAGGTAACCTGTAAGTC-3′ (SEQ ID NO: 4: “BET-022”)and flanking restriction enzyme sites (BspEI and Xho I) useful forcloning into plasmid pMJB107 sites. The resulting DNA is refererred toas PCR fragment A.

An efficient signal sequence from the human vascular cell adhesionmolecule-1 (VCAM-1) signal sequence and a six histidine tag wereintroduced into the final construct from a second DNA fragment createdfrom pDSW247 (fragment B). Plasmid pDSW247 is a derivative of pCEP4(Invitrogen, Carlsbad, Calif.) from which the EBNA-1 gene has beendeleted, and which carries the VCAM-1 signal sequence (VCAMss) fusedupstream and in frame with a six histidine tag. The PCR primers thatwere used to generate the VCAMss-1/histidine tag cassette moiety wereKID-369

(5′ PCR primer 5′-AGCTTCCGGGGGCCATCATCATCATCATCATAGCT-3′: SEQ ID NO: 5)and KTD-421 (3′ PCR primer 5′-CCGGAGCTATGATGATGATGATGATGGCCCCCGGA-3′:SEQ ID NO: 6)incorporating flanking restriction enzyme cleavage sites (NotI andBspEI) that allowed excision of the fragment B DNA.

To create a plasmid vector that carried the VCAM-1 signal sequence, histag and interferon-beta gene we performed a three-way ligation using gelpurified DNA fragments from plasmid vector pMJB 107 (NotI and XhoIcleaved), PCR fragment A (BspEI and XhoI cleaved) and fragment B (NotIand BspEI cleaved). The ligated plasmid was used to transform eitherJA221 or XL1-Blue E. coli cells and ampicillin resistant colonies werepicked and tested for inserts by restriction map analysis. Maxiprep DNAwas made and the sequence of the insert was verified by DNA sequencing.The resulting construct was called pCMG260.

-   2. Creation of alanine substitution mutants of human interferon-beta    in pCMG260    The plasmid pCMG260 was used as a template for multiple rounds of    mutagenesis (U.S.E. Site Directed Mutagenesis Kit    (Boehringer-Mannheim), which introduced unique restriction cleavage    sites into positions along the IFN-beta protein coding sequence but    did not change the resulting sequence of the protein. The    mutagenized plasmids were used to transform either the JA221 or    XL1-Blue strains of E. coli and recombinant colonies selected for    chloramphenicol resistance. Chloramphenicol resistant colonies were    further tested for the presence of the desired unique restriction    enzyme site by DNA restriction mapping analysis. The resulting    IFN-beta plasmid, pCMG275.8, contained the full set of unique    restriction enzyme cleavage sites and the DNA sequence of the gene    was verified. The full DNA sequence (SEQ ID NO: 1) of the modified,    his-tagged interferon beta gene, together with the protein coding    sequence (SEQ ID NO: 2), are given in FIG. 10.

The full set of alanine substitution mutations are depicted in Table 1(below). The names of the mutants specify the structural regions(helices and loops) in which the mutations were introduced. The entirepanel of alanine (serine) substitutions results in mutation of 65 of the165 amino acids of human IFN-beta.

The panel of mutants was created from pCMG275.8 by replacing segments ofDNA between the unique restriction sites with synthetic oligonucleotideduplexes, which carried the genetic coding information depicted in Table2 (see below). To create the various alanine substitution mutantplasmids gel purified pCMG275.8 vector (cleaved with the appropriaterestriction enzyme, as indicated on the list below for each IFN-betastructural region) and oligonucleotide duplexes (coding strand sequencesare shown in Table 2) were ligated together. The ligation mixtures wereused to transform the JA221 strain of E. coli and recombinant coloniesselected for ampicillin resistance. Ampicillin resistant colonies weretested for the presence of the insertion of the mutations by screeningfor appropriate restriction enzyme sites. For two mutants (A2 and CD2),the cloning strategy entailed using two duplexes of syntheticoligonucleotides (shown in Table 2), which carry complementaryoverhanging ends to allow them to ligate to each other and thevector-IFN-beta backbone in a three-way ligation. The following listillustrates the sites which were used to clone the mutatedoligonucleotides from Table 2. The cloning scheme (subsection B) showsthe positions of these unique sites on the interferon beta gene.

TABLE 1 Positions of alanine substitution mutations of ^(HU)IFN-β1        10        20        30        40        50 |.  ..  .|.   ....| ..    ..|. ..  .. .|  . .....|... IFN-βMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKE A1-A-AA--A--A------------------------------------------ A2--------------AA-AA--AA------------------------------ AB1---------------------------AAA-AA-------------------- AB2-----------------------------------AA-A--A----------- AB3--------------------------------------------AAAAA-AAA _(|———————)helixA_(—————————||————————)AB loop_(————————————|)      60        70        80        90       100       |   .  ..| ...     |..  ..  .| .   .  .|. ..  ... IFN-βDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVEEKLEKE B1----------A--AS----------------------------------------- B2-----------------AAA------------------------------------ C1---------------------------AS--AA--S-------------------- C2--------------------------------------A---A--AA--------- CD1-------------------------------------------------AA--AAA_(|————————)helix B_(—||—————————————————————————||—)CD loop_(——)110      120       130       140       150       160 |.. .  . .|       .|.. . .. .|.      . | .  .  .| IFN-βDFTRGALMSSHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYRINRLTGYLRN CD2AA-A--A--A----------------------------------------------- D------------------A-AA--A-------------------------------- DE1--------------------------AA----------------------------- DE2-----------------------------AA-------------------------- E--------------------------------------A---A--A--A-------- CDloop_(—||————)helix D_(————|     |——————)helix E_(———————————|)The line designated IFN-β shows the wild type human IFN-β sequence.Alanine or serine substitutions of the IFN-β residues are shown for eachof the mutants and dashes, below relevant regions, indicate wild typesequences. The helices and loop structures are indicated as solid linesbelow the mutants. The DE loop spans the gap between the D and Ehelices. Two additional alanine substitution mutants (H93A, H97A andH121A) were generated and analyzed in antiviral assays to assess theeffects of mutating these histidines, which chelate zinc in the crustalstructure dimer. Both of these mutants retained full wild type activityin antiviral assays, suggesting that zinc-mediated dimer formation isnot important for IFN-β activity.

TABLE 2 A1 SEQ ID NO: 7CCGGAGACGATGATGACAAGATGGCTTACGCCGCTCTTGGAOCCCTACAAGC BET-053TTCTAGCAATTTTCAGTGTCAGAAGCTCCTGTGGC A2 SEQ ID NO: 8CATCTAGCAATGCTGCCTGTGCTGCCCTCCTGGCTGCCTTGAATGGGAGGCTT BET-039 GAATACTSEQ ID NO: 9 GCCTCAAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCABET-041 AB SEQ ID NO: 10AATTGAATGGGAGGGCTGCAGCTTGCGCTGCAGACAGGATGAACTTTGACATCC 1 BET-080CTGAGGAGATTAAGCAGCTGCA AB SEQ ID NO: 11AATTGAATGGGAGGCTTGAATACTGCCTCAAGGACAGGGCTGCATTTGCTATC 2 BET-082CCTGCAGAGATTAAGCAGCTGCA AB SEQ ID NO: 12AATTGAATGGGAGGCTTGAATACTGCCTCAAGGACAGGATGAACTTTGACA 3 BET-084 SEQ ID NO:13 TCCCTGAGGAGATTGCTGCAGCTGCAGCTTTCGCTGCAGCTGA BET-086 B1 SEQ ID NO: 14CGCCGCGTTGACCATCTATGAGATGCTCGCTAACATCGCTAGCATTTTCA BET-110GACAAGATTCATCTAGCACTGGCTGGAA B2 SEQ ID NO: 15CGCCGCATTGACCATCTATGAGATGCTCCAGAACATCTTTGCTATTTTCGC BET-112TGCAGCTTCATCTAGCACTGGCTGGAA C1 SEQ ID NO: 16GCAATGCTTCAATTGTTGCTGCACTCCTGAGCAATGTCTATCATCAGATAA BET-114ACCATCTGAACACACTTCTAG C2 SEQ ID NO: 17GGAATGACACCATTGTTGAGAACCTCCTGGCTAATGTCGCTCATCAGATAG BET-092CACATCTCCCTCCAGTTCTAG CD SEQ ID NO: 18CTAGCTGCAAAACTGGCTGCAGCTGATTTCACCAGGGGAAAACT 1 BET- 094 CD SEQ ID NO: 19CTAGAAGAAAAACTGGAGAAAGAAGCAGCTACCGCTGGAAAAGCAATGA 2 BET- 096GCGCGCTGCACCTGAAAAGA SEQ ID NO: 20TATTATGGGAGGATTCTGCATTACCTGAAGGCCAAGGAGTACTCACACTGT BET-106 D1 SEQ IDNO: 21 CATGAGCAGTCTGCACCTGAAAAGATATTATCGGGCAATTGCTGCATACCTG BET-108GCAGCCAACGACTACTCACACTGT DE SEQ ID NO: 22CATGAGCAGTCTGCACCTGAAAAGATATTATGGGAGGATTCTGCATTACCTG 1 BET-116AAGGCCGCTGCATACTCACACTGTGCCTGGACGAT DE SEQ ID NO: 23CATGAGCAGTCTGCACCTGAAAAGATATTATGGGAGGATTCTGCATTACCTGA 2 BET-118AGGCAAAGGAGTACGCTGCATGTGCCTGGACGAT E1 SEQ ID NO: 24CGTCAGAGCTGAATCCTAGCAAACTTTGCATTCATTGCAAGACTTACAG BET-104

-   B. Construction of EBNA 293 Expression Plasmids

The wild type and mutant IFN-beta genes, fused to the VCAM-1 signalsequence, his tag and enterokinase cleavage site, were gel purified as761 base pair NotI and BamHI restriction fragments. The purified geneswere subcloned into NotI and BamHI cleaved plasmid vector pDSW247, asdepicted in the schematic. Plasmid pDSW247 is an expression vector fortransient expression of protein in human EBNA 293 kidney cells(Invitrogen, Carlsbad, Calif.). It contains the cytomegalovirus earlygene promoter and EBV regulatory elements which are required for highlevel gene expression in that system, as well as selectable markers forE. coli (ampicillin resistance) and EBNA 293 cells (hygromycinresistance) as seen in the cloning strategy schematic (below). Theligated plasmids were used to transform either JA221 or XL1-Blue E. colicells and ampicillin resistant colonies were picked and tested forinserts by restriction map analysis. Maxiprep DNA was made and thesequence of the inserts was verified by DNA sequencing. Positive clonesdisplaying the desired mutagenized sequences were used to transfecthuman EBNA 293 kidney cells as described below.

-   C. Expression and Quantitation of IFN-beta-1a Alanine Substitution    Mutants

The human EBNA 293 cells (Invitrogen, Carlsbad, Calif., Chittenden, T.(1989) J. Virol. 63: 3016-3025) were maintained as subconfluent culturesin Dulbecco's Modified Eagle's media supplemented with 10% fetal bovineserum, 2 mM glutamine and 250 μg/ml Geneticin (Life Technologies,Gaithersburg, Md.). The pDSW247 expression plasmids were transientlytransfected into EBNA 293 cells using the lipofectamine protocol(Gibco/BRL, Life Technologies). Conditioned media was harvested 3-4 daysposttransfection, cell debris was removed by centrifugation, and thehis-IFN-beta concentration was quantitated by ELISA.

The ELISA assay was performed using polyclonal rabbit antibodies(protein A purified IgG, antibodies had been raised to purified humanIFN-beta-1a) to coat 96-well ELISA plates and a biotinylated form of thesame polyclonal rabbit IgG was used as a secondary reagent to allowinterferon detection using streptavidin-linked horseradish peroxidase(HRP: Jackson ImmunoResearch, W. Grove, Pa.). A dilution series ofinterferon-beta-1a was used to generate standard concentration curves.The his-IFN-beta containing conditioned media from the EBNAtransfectants were diluted to obtain samples with concentrations rangingbetween 10 ng/ml and 0.3 ng/ml in the ELISA assay. To confirm theconcentrations of the IFN-beta in media determined by ELISA, westernblot analysis was performed. Reduced culture supernatants andIFN-beta-1a standards were subjected to SDS-PAGE on 10-20% gradient gels(Novex, San Diego, Calif.) and blotted onto PDVF membranes.Immunoreactive bands were detected with a rabbit polyclonalanti-IFN-beta-1a antiserum (#447, Biogen, Inc., a second antiserum thathad been raised against IFN-beta-1a), followed by treatment withHRP-linked donkey anti-rabbit IgG (Jackson ImmunoResearch).

-   D. Assessing the Interferon-beta Mutants for Receptor Binding

The receptor binding properties of the Interferon-beta mutants describedin C were assessed using two different binding assays. One assaymeasured binding of the interferon-beta mutants to a fusion protein,IFNAR2/Ig, comprising the extracellular domain of the human IFNAR2receptor chain fused to part of the constant region of a human IgG.IFNAR2-Fc was expressed in chinese hamster ovary (CHO) cells andpurified by protein A sepharose affinity chromatography according to theinstructions of the manufacturer (Pierce Chem. Co., Rockford, Ill.,catalog #20334). The binding of interferon-beta mutants to IFNAR2-Fc wasmeasured in an ELISA format assay. ELISA plates were prepared by coatingflat-bottomed 96 well plates overnight at 4° C. with 50 μl/well of mouseanti-human IgG1 monoclonal antibody (CDG5-AA9, Biogen, Inc.) at 10 μg/mlin coating buffer (50 mM NaHCO₃, 0.2 mM MgCl₂, 0.2 mM CaCl₂, pH 9.6).Plates were washed twice with PBS containing 0.05% Tween-20, and blockedwith 0.5% non-fat dry milk in PBS for 1 hour at room temperature. Aftertwo more washes, 50 μl of 1 μg/ml IFNAR2-Fc in 0.5% milk in PBScontaining 0.05% Tween-20 was added to each well and incubated for 1hour at room temperature, and the plates were then washed twice more.Binding of the interferon-beta mutants to IFNAR2-Fc was measured byadding 50 μl/well mutant interferon-beta in conditioned media, seriallydiluted in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with10% fetal bovine serum, and incubating for 2 hours at 4° C. Dilutions ofinterferon-beta mutant typically ranged from approximately 1 μM down to10 pM. After washing, interferon-beta bound to the plates was detectedby adding 50 μl/well of a cocktail consisting of a 1:1000 dilution of arabbit polyclonal anti-interferon antibody (#447) plus horseradishperoxidase (HRP)-labelled donkey anti-rabbit IgG (JacksonImmunoResearch), and incubating for 15 minutes at 4° C. After twowashes, HRP substrate was added, and the plate was incubated at 4° C.before being read on an ELISA plate reader at an absorbance of 450 nm.Data were plotted as absorbance versus the concentration of mutantinterferon-beta, and the affinity for the binding of the mutantinterferon-beta to IFNAR2-Fc was determined by fitting the data to asimple hyperbolic binding equation. Results from these analyses areshown in FIG. 1, in which the binding affinity for each mutant,determined at least three independent experiments, is expressed as apercentage of that measured for His₆-wild-type interferon-beta-1a.

A second receptor binding assay was used to measure the affinity withwhich the interferon-beta mutants bound to Daudi cells expressing bothreceptor chains, IFNAR1 and IFNAR2, which together comprise the receptorfor interferon-beta. This FACS-based assay used a blocking monoclonalantibody directed against the extracellular domain of IFNAR1, EA12(Biogen, Inc.), to distinguish unoccupied (free) receptor from receptorto which interferon-beta was bound. Daudi cells (20 μl at 2.5×10⁷cells/ml) were placed in 96-well V-bottom ELISA plates, and incubatedfor 1 hour at 4° C. with various concentrations of interferon-betamutant (20 μl in FACS buffer; 5% FBS, 0.1% NaN₃ in PBS). Desirableserial dilutions of interferon-beta mutants ranged from 0.5 μM down to0.5 pM. To each well was added 100 ng of biotinylated murine anti-IFNAR1monoclonal antibody EA12 (10 μl), and the plates incubated for anadditional 2 minutes at room temperature before being washed twice withFACS buffer (4° C.). The cells were then incubated for 30 minutes at 4°C. with 50 μl/well of a 1:200 dilution of R-Phycoerythrin-conjugatedstreptavidin (Jackson ImmunoResearch), washed twice in FACS buffer,resuspended in 300 μl FACS buffer containing 0.5% paraformaldehyde, andtransferred into 12×75 mm polystyrene tubes (Falcon 2052). The sampleswere then analyzed by flow cytometry on a FACScan (Becton Dickinson).Data were plotted as mean channel fluorescence intensity (MFCI) versusthe concentration of interferon-beta mutant; binding affinities weredefined as the concentration of interferon-beta mutant giving 50%inhibition of antibody staining. Each mutant was tested multiple times.FIG. 2 shows the receptor binding affinities for each interferon-betamutant, determined by this method, expressed as a percentage of theaffinity measured for His₆-wild-type interferon-beta-1a in eachexperiment.

-   E. Assessing the Interferon-beta Mutants for Function

The interferon-beta mutants were also tested for functional activityusing in vitro assays for antiviral activity and for the ability of theinterferon-beta to inhibit cell proliferation. A minimum of threeantiviral assays, each with triplicate data points, were performed oneach mutant. His₆-wild-type interferon-beta-1a was included as areference in every experiment. The antiviral assays were performed bytreating A549 human lung carcinoma cells (ATCC CCL 185) overnight with2-fold serial dilutions of mutant interferon-beta at concentrations thatspanned the range between full antiviral protection and no protectionfrom viral cell killing. The following day, the cells were challengedfor two days with encephalomyocarditis virus (ECMV) at a dilution thatresulted in complete cell killing in the absence of interferon. Plateswere then developed with the metabolic dye MTT(2,3-bis[2-Methoxy-4-nitro-5-sulfo-phenyl]-2H-tetrazolium-5-carboxyanilide)(M-5655, Sigma, St. Louis, Mo.). A stock solution of MTT was prepared at5 mg/ml in PBS and sterile filtered, and 50 μl of this solution wasdiluted into cell cultures (100 μl per well). Following incubation atroom temperature for 30-60 minutes, the MTT/media solution wasdiscarded, cells were washed with 100 μl PBS, and finally themetabolized dye was solubilized in 100 μl 1.2N hydrochloric acid in 90%isopropanol. Viable cells (as evidenced by the presence of the dye) werequantified by absorbance at 450 nm. Data were analyzed by plottingabsorbance against the concentration interferon-beta mutant, and theactivity of each mutant was defined as the concentration at which 50% ofthe cells were killed. FIG. 3 shows the activity of each mutantexpressed as a percentage of the activity measured for histagged-wild-type interferon-beta-1a in each experiment.

Interferon-beta mutants were also assessed for function in anantiproliferation assay. Human Daudi Burkitt's lymphoma cells (ATCC #CCL 213) were seeded at 2×10⁵ cells/ml in RPMI 1620 supplemented with10% defined fetal calf serum (Hyclone, Logan Utah), and 2 mML-glutamine. Each well also contained a given concentration ofinterferon-beta mutant in a final total volume of 100 μl of medium perwell; the interferon-beta concentrations used were chosen to span therange from maximal inhibition of Daudi cell proliferation to noinhibition (i.e. full proliferation). Duplicate experimental points wereused for each concentration of interferon-beta mutant tested, and aduplicate set of untreated cells was included in all experiments. Cellswere incubated for two days at 37° C. in 5% CO₂ incubators, after which1 μCi per well of tritiated thymidine ((methyl-³H) thymidine, AmershamTRK758) in 50 μl medium was added to each well, and incubated for afurther 4 h. Cells were harvested using a LKB plate harvester, andincorporation of tritiated thymidine was measured using a LKB beta platereader. Duplicate experimental values were averaged and the standarddeviations determined. Data were plotted as mean counts per minuteversus the concentration of interferon-beta mutant, and the activity ofeach mutant was defined as the concentration required to give 50% of themaximal observed growth inhibition. Multiple assays for each mutant wereperformed. FIG. 4 shows the results expressed as a percentage of theactivity found for his tagged-wild-type interferon-beta-1a in eachexperiment.

-   F. Properties of the Interferon-Beta Mutants

Histidine tagged-wild-type interferon-beta-1a was found to haveactivities in the antiviral and antiproliferation assays that were eachabout 3-fold lower than the corresponding activities found for untaggedwild-type interferon-beta-1a. Because all of the interferon-beta mutantsA1-E contain the identical his tag sequence at their N-termini, theeffects of the mutations on the properties of the molecule weredetermined by comparing the activities of these mutants in theantiviral, antiproliferation and binding assays to the activity observedfor his tagged-wild-type interferon-beta-1a. In so doing, we assume thatvariations in the activities of mutants A1-E, compared to histagged-wild-type interferon-beta-1a, are qualitatively andquantitatively about the same as the effects that these same mutationswould have in the absence of the N-terminal his tag. The equivalentassumption for tagged or fusion constructs of other soluble cytokines iscommonly held to be true by practitioners of the technique of alaninescanning mutagenesis, especially when the in vitro functional activityof the tagged or fusion construct is close to that of the wild-typecytokine as is the case here. See, for example, Pearce K. H. Jr, et al.,J. Biol. Chem. 272:20595-20602 (1997) and Jones J. T., et al., J. Biol.Chem. 273:11667-11674 (1998)

The data shown in FIGS. 1-4 suggests three types of effects that werecaused by the targeted mutagenesis. These effects may be advantageousfor interferon drug development under certain circumstances. The threetypes of effect are as follows: (a) mutants with higher antiviralactivity than that of wild-type interferon-beta-1a (e.g. mutant C1); (b)mutants which display activity in both antiviral and antiproliferationassays, but for which antiproliferation activity is disproportionatelylow with respect to antiviral activity, compared to wild-typeinterferon-beta-1a (e.g., mutants C1, D and DE1); and (c) functionalantagonists (e.g., A1, B2, CD2 and DE1), which show antiviral andantiproliferative activities that are disproportionately low withrespect to receptor binding, compared to wild-type interferon-beta-1a.It can be seen that some mutants fall into more than one class. Theseclasses are reviewed below. While we have characterized these classes ofmutants with respect to those examples listed, it should be appreciatedthat other mutations in these regions may result in similar, or evenenhanced effects on activity:

a) Mutant C1 possesses antiviral activity that is approximately 6-foldgreater than that of wild-type his tagged-interferon-beta-1a. Thismutant and others of this type are predicted to be useful in reducingthe amount of interferon-beta that must be administered to achieve agiven level of antiviral effect. Lowering the amount of administeredprotein is expected to reduce the immunogenicity of the protein and mayalso reduce side-effects from non-mechanism-based toxicities. Mutationsin this class are predicted to be advantageous in situations where thetherapeutic benefit of interferon-beta administration results from itsantiviral effects, and where antiproliferative effects contribute totoxicity or to unwanted side-effects.

(b) The relative activities (% wild type) of the alanine substitutedmutants in antiviral and antiproliferation assay are compared in FIG. 5.Coordinately changed activities (i.e. antiviral and antiproliferationactivities that differ by the same factor from the activities of thewild-type his tagged-interferon-beta-1a) are seen in most mutants (thoselying on the diagonal line). However, several mutants show greateralterations in activity in one assay relative to the other, compared towild-type his tagged-interferon-beta-1a, as evidenced by displacementfrom the diagonal. Three such mutants are shown in the Table 3 below.Mutant C1 shows antiviral activity that is ˜6-fold higher than that ofwild-type his tagged-interferon-beta-1a, but its activity in theantiproliferation assay is similar to that of wild-type. Mutant C1 thushas antiviral activity that is enhanced by a factor of 5.2 over itsantiproliferation activity, relative to wild-type histagged-interferon-beta-1a. Similarly, mutant D displays 65% of wild typeactivity in the antiviral assay, but only 20% of wild-type activity inthe antiproliferation assay, and thus has antiviral activity that isenhanced 3.4-fold over its antiproliferation activity compared to wildtype. Mutant DE1 displays 26% of wild type activity in the antiviralassay but only 8.5% in the antiproliferation assay, and thus hasantiviral activity that is enhanced 3.0-fold over its antiproliferationactivity compared to wild-type his tagged-interferon-beta-1a. Whenadministered at a concentration sufficient to achieve a desired level ofantiviral activity, these mutant proteins will show substantially lowerlevels of antiproliferative activity than the wild-type protein.Mutations in this class, like those in class (a), are predicted to beadvantageous in situations where the therapeutic benefit ofinterferon-beta administration results from its antiviral effects, andwhere antiproliferative effects contribute to toxicity or to unwantedside-effects.

TABLE 3 Antiviral Antiproliferative Activity (AV) (AP) Activity AV/AMutant (% wild type) (% wild type) P C1 571 109 5.2 D  65 19 3.4 DE1  268.5 3.0

(c) Mutants with antiviral and antiproliferative activities that are lowwith respect to receptor binding, as compared to wild-type histagged-interferon-beta-1a (see Table 4 below). Mutant A1 displaysantiviral and antiproliferative activities that are 2.0-fold and1.8-fold higher than that observed for wild-type histagged-interferon-beta-1a, but binds to the cognate receptor on Daudicells with an affinity that is 29-fold higher than wild-type. Thebinding of this mutant to the IFN-beta receptor is thus enhancedapproximately 15-fold compared to the antiviral and antproliferationactivities of the protein. Similarly, mutants B2, CD2 and DE1 showenhancements of binding over antiviral activity of 4.6-, 4.6- and18-fold, respectively, and over antiproliferation activity of 3.5-, 15-and 54-fold. These proteins are predicted to be useful as functionalantagonists of the activity of endogenous IFN-beta, and possibly ofother endogenous Type I interferons, because they have the ability tobind to and occupy the receptor, and yet generate only a small fractionof the functional response in the target cells that would be seen withwild type IFN-beta.

TABLE 4 Antiviral Activity Antiprolifera- Cell Binding Bind- Mu- (AV) (%tive Activity Activity (% ing/ tant wt) (AP) (% wt) wt) Binding/AV AP A1200 180 2900 15 16 B2 7.1 9.2 33 4.6 3.5 CD 150 46 690 4.6 15 2 DE 268.5 460 18 54 1

-   G. Mutein Relationship to Three Dimensional Structure of Interferon

While published crystal structures for a non-glycosylated form of murineinterferon beta (T. Senda, S. Saitoh and Y. Mitsui. Refined CrystalStructure of Recombinant Murine Interferon-β at 2.15 Å Resolution. J.Mol. Biol. 253: 187-207 (1995)) and for human interferon alpha-2b (R.Radhakrishnan, L. J. Walter, A. Hruza, P. Reichert, P. P Trotta, T. L.Nagabhushan and M. R. Walter. Zinc Mediated Dimer of HumanInterferon-α2b Revealed by X-ray Crystallography. Structure. 4:1453-1463 (1996)) had provided models for the polypeptide backbone ofhuman interferon beta, we have recently solved the structure forinterferon-beta-1a in its glycosylated state (M. Karpusas, M. Nolte, C.B. Benton, W. Meier, W. N. Lipscomb, and S. E Goelz. The CrystalStructure of Human Interferon-β at 2.2 Å resolution. Proc. Natl. Acad.Sci. USA 94: 11813-11818 (1997)).

The results of our mutational analyses can be summarized with respect tothe 3D-structure of interferon-beta-1a (not presented here). Certainmutatations created a reduction in activity (2 to >5 fold reduced). Themutated regions correspond to the substitutions given in Tables 1 and 2.Residues important for antiviral and antiproliferation activity arelocalized to the lower half of the IFN-beta-1a molecule (Panel a and b).Mutations in the upper half of the molecule, where the amino and carboxytermini are positioned, had no effect on biological activities orreceptor binding.

Mutations in the A2 helix, AB, AB2 loop and E helix are most significantin their effect on function and resulted in a dramatic reduction in bothactivity and cell surface receptor binding. This region (A2 helix, AB&AB2 loop and E helix) corresponds to the IFNAR2 binding site, since noneof these mutants bound IFNAR/Fc in our assay.

While those mutations that were important for IFNAR2 binding alsoaffected cell binding, cell surface binding properties are alsoinfluenced by residues in other regions of the molecule (B1 helix, C2helix). It can be seen in the 3-D models (not presented here) depictingthe effects of the alanine substitution mutants that the N-terminal,C-terminal and the glycosylated C helix regions of the IFN-beta-1amolecule do not lie within the receptor binding site. Mutations in theseregions did not reduce biological activity or reduce cell surfacereceptor binding.

EXAMPLE 2 Preparation and Characterization of ConjugatedInterferon-beta-1a

A. Preparation of PEGylated Interferon.

Nonformulated interferon-beta-1a (sold as AVONEX®) bulk intermediate at250 Tg/ml in 100 mM sodium phosphate pH 7.2, 200 mM NaCl) was dilutedwith an equal volume of 100 mM MES pH 5.0 and the pH was adjusted to 5.0with HCl. The sample was loaded onto an SP-Sepharose® FF column(Pharmacia, Piscataway, N.J.) at 6 mg interferon-beta-1a/ml resin. Thecolumn was washed with 5 mM sodium phosphate pH 5.5, 75 mM NaCl, and theproduct was eluted with 30 mM sodium phosphate pH 6.0, 600 mM NaCl.Elution fractions were analyzed for their absorbance values at 280 nmand the concentration of interferon in the samples estimated from theabsorbance using an extinction coefficient of 1.51 for a 1 mg/mlsolution.

To a 1 mg/ml solution of the interferon-beta-1a from the SP eluate, 0.5M sodium phosphate pH 6.0 was added to 50 mM, sodium cyanoborohydride(Aldrich, Milwaukee, Wis.) was added to 5 mM, and 20K PEG aldehyde(Shearwater Polymers, Huntsville, Ala.) was added to 5 mg/ml. The samplewas incubated at room temperature for 20 hours. The pegylated interferonwas purified from reaction products by sequential chromatography stepson a Superose® 6 FPLC sizing column (Pharmacia) with 5 mM sodiumphosphate pH 5.5, 150 mM NaCl as the mobile phase and SP-Sepharose® FF.The sizing column resulted in base line separation of modified andunmodified interferon beta (chromatograph not presented here). ThePEG-interferon beta-containing elution pool from gel filtration wasdiluted 1:1 with water and loaded at 2 mg interferon beta/ml resin ontoan SP-Sepharose® column. The column was washed with 5 mM sodiumphosphate pH 5.5, 75 mM NaCl and then the pegylated interferon beta waseluted from the column with 5 mM sodium phosphate pH 5.5, 800 mM NaCl.Elution fractions were analyzed for protein content by absorbance at 280nm. The pegylated interferon concentration is reported in interferonequivalents as the PEG moiety did not contribute to absorbance at 280nm.

B. Biochemical Characterization of PEGylated Interferon.

Samples were analyzed for extent of modification by SDS-PAGE (gel notpresented here). Addition of a single PEG resulted in a shift in theapparent mass of interferon from 20 kDa to 55 kDa which was readilyapparent upon analysis. In the pegylated sample there was no evidence ofunmodified interferon-beta-1a nor of higher mass forms resulting fromthe presence of additional PEG groups. The presence of a single PEG wasverified by MALDI mass spectrometry. The specificity of the pegylationreaction was evaluated by peptide mapping. 20 Tg aliquots of pegylated,and unmodified interferon-beta-1a as a control, in 240 TL of 200 mM TrisHCl pH 9.0, 1 mM EDTA were digested with 1.5 Tg of lysyl endoproteinasefrom Achromobacter (Wako Bioproducts, Richmond, Va.) for 3-4 hours at27° C. 200 mg of guanidine HCl was added to each sample and the cleavageproducts were fractionated on a Vydac C₄ column (0.46×25 cm) using a 30min gradient from 0 to 70% acetonitrile, in 0.1% TFA at a flow rate of1.4 ml/min. The column effluent was monitored for absorbance at 214 nm.

Results from the analysis are shown in FIG. 6. All of the predictedpeptides from the endoproteinase Lys-C digest of interferon-beta-1a havebeen identified by N-terminal sequencing and mass spectrometry and ofthese, only the peptide that contains the N-terminus of interferon (AP8)was altered by the modification as evident by its disappearance from themap. The mapping data therefore indicate that the PEG moiety isspecifically attached to this peptide. The data further indicate thatthe PEG modification is targeted at the N-terminus of the protein sinceonly the N-terminal modification would result in the specific loss ofthis peptide.

Additional evidence for this conclusion was obtained by isolating thePEGylated N-terminal peptide from the endoproteinase Lys-C digest,digesting the peptide further with cyanogen bromide (CNBr) andsubjecting this sample to matrix-assisted laser desorption ionizationpost source decay (MALDI PSD) sequence analysis. CNBr digestion of theN-terminal peptide will further cleave this peptide into two fragments,the terminal methionine (M1) containing the PEG moiety and SYNLLGFLQR(residues 2-11 in the mature interferon beta sequence) Sequence analysisidentified the unmodified peptide SYNLLGFLQR, which was the predictedoutcome of this treatment.

The antiviral activity of interferon-beta-1a samples was tested on humanlung carcinoma cells (A549 cells) that had been exposed toencephalomyocarditis (EMC) virus using the procedures involving MTTstaining outlined above. Briefly, A549 cells were pretreated for 24hours with interferon-beta-1a or PEG-modified interferon-beta-1a (4000,2000, 1000, 500, 250, 125, 75, 62.5, 31.25, 50, 33.3, 22.2, 14.8, 9.9,6.6, 4.39 pg/ml) prior to challenge with virus. The assay was performedusing duplicate data points for each interferon-beta-1a concentration.The standard deviations are shown as error bars in FIG. 7. Theconcentration of interferon-beta-1a (formulated or bulk) which offered50% viral killing (the “50% cytopathic effect”) (50% maximum OD₄₅₀) wasabout 11 pg/ml and the 50% cytopathic effect for PEG modifiedinterferon-beta-1a was about 11 pg/ml. Thus, PEG conjugation did notalter the antiviral activity of interferon-beta-1a. In this assay, weroutinely find that the specific activity of interferon-beta-1a is about10 times greater than the specific activity of interferon-beta-1b andtherefore PEGylated interferon-beta-1a is significantly more active thanany interferon-beta-1b product.

Interferon-beta-1a was also PEGylated with a 5K PEG-aldehyde moiety thatwas purchased from Fluka, Inc. (Cat. No. 75936, Ronkonkoma, N.Y.)following the same protocol described for modification with 20K PEGaldehyde except that the reaction contained 2 mg/ml of the 5K PEG.Modification with the 5K PEG was also highly specific for the N-terminusand did not alter the antiviral activity of interferon-beta-1a. Like the20K adduct, the 5K PEG inteferon-beta-1a was indistinguishable from theunmodified interferon-beta-1a in the antiviral assay.

EXAMPLE 3 PEGylation Protects Interferon-beta-1a from Stress-inducedAggregation

Aggregation of interferon beta has a deleterious effect on activity.Previously, we have shown that glycosylation has a dramatic effect onstability of interferon-beta-1a versus nonglycosylated forms ofinterferon beta and inferred that glycosylation contributes to thehigher specific activity of interferon-beta-1a (Runkel L. et al, Pharm.Res. 15: 641-649). To investigate whether conjugation with apolyalkylene glycol polymer might further stabilize interferon beta, wesubjected the PEGylated interferon-beta-1a to thermal stress using thefollowing protocol:

Thermal denaturation was carried out using a CARY 3 UV-visiblespectrophotometer fitted with a computer controlled, thermolectricallyheated cuvette holder. Solutions of interferon-beta-1a in 20 mM HEPESpH7.5, 20 mM NaCl were equilibrated at 25° C. in a 1 ml cuvette. Thetemperature of the cuvette holder was then ramped from 25° C. to 80° C.at a rate of 2° C./min, and the denaturation of the protein followed bycontinuous monitoring of absorbance at 280 nm. The mid-point of thecooperative unfolding event, Tm, was obtained from the melting curves bydetermining the temperature at which the measured absorbance was mid-waybetween the values defined by lines extrapolated from the linear regionson each side of the cooperative unfolding transitions.

Results from this analysis are shown in FIG. 8. Whereas thenon-PEGylated-interferon-beta-1a denatured and aggregated with a 50%point of transition at 60° C., there was no evidence of aggregation ofthe PEGylated interferon even at 80° C. In an independent analysis, weextended the thermal stress treatment to 95° C. and even at this moreelevated temperature, we saw no evidence for aggregation. Thus,conjugation with this polyethylene glycol polymer has a profound andbeneficial effect on the stability of the protein. Similar stabilizationwas seen with modified interferon-beta-1a containing the 20K and 5K PEG.

EXAMPLE 4 Measurement of Interferon-beta-1a Antiviral Activity in thePlasma of Mice Treated with Interferon-beta-1a and PEGylatedInterferon-beta-1a

Mice (C57Bl/6) are injected i.v. through the tail vein with either50,000 Units of interferon-beta-1a or 50,000 Units of PEGylatedinterferon-beta-1a containing the 20K PEG or an equal volume ofphosphate buffer given as a control. Blood from these mice is obtainedvia retro-orbital bleeds at different time points after injection(immediately, 0.25, 1, 4, 24 and 48 hours). There are at least 3 micebled at each time point. Whole blood is collected into tubes containinganticoagulant, cells are removed and the resulting plasma frozen untilthe time of assay. These plasma samples are then tested in anti-viralassays.

The plasma samples are diluted 1:10 into serum free media and passedthrough a 0.2 um syringe filter. Diluted samples are tested in antiviralassays. Samples are titrated into designated wells of a 96 well tissueculture plate containing A549 cells. Dilutions of a standardinterferon-beta-1a (10, 6.7, 4.4, 2.9, 1.3, 0.9 and 0.6 U/ml) and offour plasma samples were assayed on every plate. The A549 cells arepretreated with diluted plasma samples for 24 hours prior to challengewith EMC virus. Following a two-day incubation with virus, viable cellsare stained with a solution of MTT (at 5 mg/ml in phosphate buffer) for1 hour, washed with phosphate buffer, and solubilized with 1.2 N HCl inisopropanol. The wells were read at 450 nm. Standard curves aregenerated for each plate and used to determine the amount ofinterferon-beta-1a activity in each test sample. The activity in thesamples from the different mice are graphed against the time points inFIG. 9.

The slower loss of PEGylated interferon-beta-1a from circulation as afunction of time indicates that the half life of the PEGylated sample ismuch longer than that of the untreated interferon-beta-1a control.Whereas the control was largely cleared after 4 h, a significantfraction of the PEGylated product was detected after 48 h. Based on theinitial levels of activity in serum and those remaining after 48 h, weinfer that the half life of the PEGylated interferon is extended whencompared to the half life of unmodified interferon-beta-1a. A secondhighly significant finding from the study was that very little of thePEGylated form was lost during the distribution phase, as evidenced bythe similar high levels of activity at time 0 and after 60 min. The dataindicate that, unlike the control interferon-beta-1a, the distributionof the PEGylated product is largely limited to the vasculature.

EXAMPLE 5 Comparative Pharmacokinetics and Pharmacodynamics in PrimatesGeneral Protocols

Comparative studies are conducted with polymer-interferon-beta 1aconjugates and native interferon-beta 1a (as non formulated bulkintermediate interferon-beta-1a in sodium phosphate, and NaCl, pH 7.2)to determine their relative stability and activity in primates. In thesestudies, the pharmacokinetics and pharmacodynamics of thepolymer-interferon-beta-1a conjugate in primates is compared to that ofnative interferon-beta 1a and reasonable inferences can be extended tohumans.

Animals and Methods

Study Design

This is a parallel group, repeat dose study to evaluate the comparativepharmacokinetics and pharmacodynamics of conjugated and unconjugatedinterferon-beta-1a.

Healthy primates (preferably rhesus monkeys) are used for this study.Prior to dosing, all animals will be evaluated for signs of ill healthby a Lab Animal Veterinary on two occasions within 14 days prior to testarticle administration; one evaluation must be within 24 hours prior tothe first test article administration. Only healthy animals will receivethe test article. Evaluations will include a general physicalexamination and pre-dose blood draws for baseline clinical pathology andbaseline antibody level to interferon-beta-1a. All animals will beweighed and body temperatures will be recorded within 24 hours prior totest article administrations.

Twelve subjects are enrolled and assigned to groups to receive 1 MU/kgof interferon-beta-1a as either a PEG-interferon-beta-1a conjugate ornon-conjugated, but otherwise identical interferon-beta-1a.Administration is by either the subcutaneous (SC) or intravenous (IV)routes. All animals must be naive to interferon-beta treatment. Eachanimal will be dosed on two occasions; doses will be separated by fourweeks. The dose volume will be 1.0 mL/kg.

Blood is drawn for pharmacokinetic testing at various time intervalsfollowing each injection. Blood samples for measurements of theinterferon induced biological response marker, serum neopterin, are alsodrawn following administration of study drug.

Evaluations during the study period include clinical observationsperformed 30 minutes and 1 hour post-dose for signs of toxicitiy. Dailycageside observations will be performed and general appearance, signs oftoxicity, discomfort, and changes in behavior will be recorded. Bodyweights and body temperatures will be recorded at regular intervalsthrough 21 days post-dose.

Assay Methods

Levels of interferon beta in serum are quantitated using a cytopathiceffect (CPE) bioassay. The CPE assay measures levels ofinterferon-mediated antiviral activity. The level of antiviral activityin a sample reflects the number of molecules of active interferoncontained in that sample at the time the blood is drawn. This approachhas been the standard method to assess the pharmacokinetics ofinterferon beta. The CPE assay used in the current study detects theability of interferon beta to protect human lung carcinoma cells (A549,#CCL-185, ATCC, Rockville, Md.) from cytotoxicity due toencephalomyocarditis (EMC) virus. The cells are preincubated for 15 to20 hours with serum samples to allow the induction and synthesis ofinterferon inducible proteins that then mount an antiviral response.Afterwards EMC virus is added and incubated for a further 30 hoursbefore assessment of cytotoxicity is made using a crystal violet stain.An internal interferon beta standard as well as PEG conjugate internalstandard is tested concurrently with samples on each assay plate. Thisstandard is calibrated against a natural human fibroblast interferonreference standard (WHO Second International Standard for Interferon,Human Fibroblast, Gb-23-902-53). Each assay plate also includes cellgrowth control wells containing neither interferon beta of any kind norEMC, and virus control wells contain cells and EMC but no interferonbeta. Control plates containing the standard and samples are alsoprepared to determine the effect, if any, of the samples on cell growth.These plates are stained without the addition of virus.

Samples and standards are tested in duplicate on each of two replicateassay plates, yielding four data points per sample. The geometric meanconcentration of the four replicates is reported. The limit of detectionin this assay is 10 units (U)/ml.

Serum concentrations of neopterin are determined at the clinicalpharmacology unit using commercially available assays.

Pharmacokinetic and Statistical Methods

RstripTM software (MicroMath, Inc., Salt Lake City, Utah) is used to fitdata to pharmacokinetic models. Geometric mean concentrations areplotted by time for each group. Since assay results are expressed indilutions, geometric means are considered more appropriate thanarithmetic means. Serum interferon levels are adjusted for baselinevalues and non-detectable serum concentrations are set to 5 U/ml, whichrepresents one-half the lower limit of detection.

For IV infusion data, a two compartment IV infusion model is fit to thedetectable serum concentrations for each subject, and the SC data arefit to a two compartment injection model.

The following pharmacokinetic parameters are calculated:

-   -   (i) observed peak concentration, C_(max) (U/ml);    -   (ii) area under the curve from 0 to 48 hours, AUC using the        trapezoidal rule;    -   (iii) elimination half-life;        and, from IV infusion data (if IV is employed):    -   (iv) distribution half-life (h);    -   (v) clearance (ml/h)    -   (vi) apparent volume of distribution, Vd (L).

WinNonlin (Scientific Consulting Inc., Apex, N.C.) software is used tocalculate the elimination half-lives after SC and IM injection.

For neopterin, arithmetic means by time are presented for each group.E_(max), the maximum change from baseline, is calculated. C_(max), AUCand E_(max) are submitted to a one-way analysis of variance to comparedosing groups. C_(max) and AUC are logarithmically transformed prior toanalysis; geometric means are reported.

EXAMPLE 6 Comparative Evaluation of PEGylated Interferon beta-1a andInterferon-beta-1a Pharmacokinetics in Rhesus Monkeys

Materials and Methods

Interferon beta-1a or PEGylated IFN beta-1a were administered to rhesusmonkeys on day 1 and again on day 29 by the intravenous (IV) orsubcutaneous (SC) routes as described in the general protocol of Example5. On day 1, six monkeys received IFN beta-1a (3 per route) and anothersix monkeys received PEGylated IFN beta-1a (3 per route). On day 29, thedoses were repeated. The IV dose was administered as a slow bolusinjection into a cephalic or saphenous vein.

The SC dose was administered under the skin on the back after shavingthe injection site. Blood was collected via the femoral vein atspecified time points and allowed to clot to obtain serum. Serum wasanalyzed for levels of functional drug substances using a validatedantiviral CPE method and for serum neopterin and beta2-microglobulinlevels as pharmacodynamic measures of activity. Pharmacologicalparameters were calculated using WinNonlin version 2.0 software(Scientific Consulting Inc., Apex, N.C.).

The concentration data were analyzed by standard model-independentmethods (noncompartmental analysis) to obtain pharmacokineticparameters. Area under the curve (AUC) was calculated using thetrapezoidal rule. Statistical analyses, including arithmetic mean andstandard deviation, were performed using Microsoft Excel version 5.0software (Microsoft Corp., Redmond Wash.). Concentration values reportedas below limits of quantitation (BLQ) were not used in thepharmacokinetic analysis. Due to the fact that different computers andcomputer programs round off or truncate numbers differently, values insome tables (e.g. means, standard deviations, or individual values) maydiffer slightly from those in other tables, from individually calculateddata, or from statistical analysis data. Neither the integrity norinterpretation of the data was affected by these differences.

Results and Discussion

Within each route of administration, pegylated IFN beta-1a exhibitedhigher bioavailability (as measured by the area under the serumconcentration-time curve). In addition the pegylated IFN beta-1a had ahigher absolute bioavailability as compared to IFN beta-1a whenadministered by the SC route. We summarize the pharmacokineticparameters in Table 5. Administration of pegylated IFN beta-1a by bothIV and SC routes results in an increase in the half-life as well as theAUC of IFN beta-1a.

TABLE 5 Mean (±Std. Dev.) BG9418 Pharmacokinetic Parameters Following IVor SC (Dose 1) Administration of 1 MU/kg of IFN b-1a or Pegylated IFNB-1a to Rhesus Monkeys^(a) Formula- tion (Route of Administra- AUC CLVss tion) C_(max) T_(max) U*hr/mL (mL/kg) (mL/kg) T_(1/2) IFN B-1a 64000.083 4453 229 543 3.2 (IV) (±) (±0) (±799) (±38) (±147) (±1.4)Pegylated 10800 0.083 34373 29 250 9.5 IFN-b-1a (±3811) (±0) (±3601)(±3) (±30) (±2.1) (IV) IFN B-1a 277 5.3 4753 N/A N/A 10.0 (SC) (±75)(±1.2) (±3170) (±2.9) Pegylated 1080 3.3 42283 N/A N/A 22.0 IFN B-1a(±381) (±1.2) (±5934) (±3.4) (SC) ^(a)n = 3

Following IV administration of the first dose, the mean (±std. dev.)peak serum concentrations (Cmax) of IFN beta-1a and pegylated IFNbeta-1a were 6400 (±0) and 10800 (±3.5) U/mL, respectively. The mean(±std. dev.) AUC values were 4453 (±799) and 34373 (±3601) U*hr/mL,respectively. Following the first SC administration, the mean (±std.dev.) Cmax of IFN beta-1a and pegylated IFN beta-1a were 277 (±75) and1080 (±381) U/mL, respectively. Mean (±std. dev.) AUC values were 4753(±3170) and 44952 (±1443) U*hr/mL, respectively.

Both serum neopterin and serum beta2microglobulin levels were elevatedafter treatment with IFN-beta and pegylated IFN-beta, indicatingpharmacologic activity of the products. At the high doses of testcompounds used, there was no difference in the pharmacologic activity ofIFN beta-1a and pegylated IFN beta-1a by either route of administration(data now shown).

EXAMPLE 7 Comparative Evaluation of Pegylated Interferon beta-1a andInterferon-beta-1a Pharmacokinetics in Rats Following Various Modes ofAdministration

The purpose of this study was to determine the comparativebioavailability of interferon beta-1a and pegylated interferon beta-1aby several routes of administration.

Materials and Methods:

We used female Lewis rats (at 190 grams each) for pharmacokineticanalyses with two rats per route/formulation. The rats were jugularcannulated and either human interferon beta-1a or 5K pegylated humaninterferon beta-1a or 20K human interferon beta-1a (in a vehicleconsisting of 14 mg/ml HSA in 50 mM sodium phosphate, 100 mM NaCl, pH7.2) were admininstered intravenously, intraperitoneally, orally,subcutaneously or intratracheally. Blood was processed several timesover a 72 hour period at 0, 5 min, 15 min, 30 min, 75 min, 3 hr, 24 h,48 h and 72 h. The protocol is presented in Table 6. The cytopathiceffect (CPE) bioassay was run on the serum samples to detectinterferon-beta in the serum. The results generated with unmodifiedinterferon beta-1a and interferon beta-1a pegylated with 20K PEG arepresented in Table 7. In all cases, pegylation resulted in significantincreases in t½ and AUC.

TABLE 6

TABLE 7 Pharmacokinetic parameters following IV, SC, IP or ITadministration of Interferon beta-1a (IFN) and pegylated IFN-beta 1a(IFN-PEG) in Rats Formulation (Route of C_(max) T_(max) AUC/Dose T_(½)Administration) (U/mL) (hr) (U hr)/(mL ug) (hr) IFN (IV, 64000 0.25 30351.25 20 ug does) IFN-PEG (IV, 23970 0.08 47728 8.44 3 ug dose) IFN (SC, 2400 1.00 464.4 0.96 20 ug dose) UFN-PEG (SC,  2400 7.00 14688 11.9 3ug dose) IFN (IP, 26000 1.25 4159 1.53 20 ug dose) IFN-PEG (IP,  97001.25 52148 16.2 3 ug dose) IFN (IT,  240 1.5  70.7 1.29 15 ug dose)IFN-PEG (IT,  270 7.0  233.5 6.21 15 ug dose)

EXAMPLE 8 Anti-Angiogenic Effects of Polymer-Conjugated InterferonBeta-1a Assessment of the Ability of PEGylated Interferon-beta-1a toInhibit Endothelial Cell Proliferation In Vitro

Human venous endothelial cells (Cell Systems, Cat. #2V0-P75) and humandermal microvascular endothelial cells (Cell Systems, Cat. # 2M1-C25)are maintained in culture with CS-C Medium Kit (Cell Systems, Cat. #4Z0-500). Twenty-four hours prior to the experiment, cells aretrypsinized, and resuspended in assay medium, 90% M199 and 10% fetalbovine serum (FBS), and are adjusted to desired cell density. Cells arethen plated onto gelatin-coated 24 or 96 well plates, either at 12,500cells/well or 2,000 cells/well, respectively.

After overnight incubation, the assay medium is replaced with freshmedium containing 20 ng/ml of human recombinant basic Fibroblast GrowthFactor (Becton Dickinson, Cat. # 40060) and various concentrations ofconjugated and unconjugated interferon-beta-1a proteins or positivecontrol (endostatin can be used as a positive control, as could anantibody to bFGF). The final volume is adjusted to 0.5 ml in the 24 wellplate or 0.2 ml in the 96 well plate.

After seventy-two hours, cells are trypsinized for Coulter counting,frozen for CyQuant fluorescense reading, or labeled with [3H] thymidine.The inhibition of endothelial cell proliferation in vitro by conjugatedand unconjugated interferon-beta 1a was comparable, indicating thatPEGylation had not interfered with the ability of the interferon tofunction in this setting.

This in vitro assay tests the human interferon-beta molecules of theinvention for effects on endothelial cell proliferation which may beindicative of anti-angiogenic effects in vivo. See O'Reilly, M. S., T.Boehm, Y. Shing, N. Fukal, G. Vasios, W. Lane, E. Flynn, J. Birkhead, B.Olsen, and J. Folkman. (1997). Endostatin: An Endogenous Inhibitor ofAngiogensis and Tumor Growth. Cell 88, 277-285.

EXAMPLE 9 In Vivo Model to Test Anti-Angiogenic and NeovascularizationEffects of Conjugated Interferon-beta-1a

A variety of models have been developed to test for the anti-angiogenicand anti-neovascularization effects of the molecules described herein.Some of these models have been described in U.S. Pat. No. 5,733,876(Mar. 31,1998: “Method of inhibiting angiogenesis) and U.S. Pat. No.5,135,919 (Aug. 4, 1992:“Method and a pharmaceutical composition for theinhibition of angiogenesis”). Other assays include the shell-lesschorioallantoic membrane (CAM) assay of S. Taylor and J. Folkman;Nature, 297, 307 (1982) and R. Crum. S. Szabo and J. Folkman; Science.230. 1375 (1985); the mouse dorsal air sac method antigiogenesis modelof Folkman, J. et al.; J. Exp. Med., 133, 275 (1971) and the rat cornealmicropocket assay of Gimbrone, M. A. Jr. et al., J. Natl. Cancer Inst.52, 413(1974) in which corneal vascularization is induced in adult malerats of the Sprague-Dawley strain (Charles River, Japan) by implanting500 ng of basic FGF (bovine, R & D Systems, Inc.) impregnated in EVA(ethylene-vinyl acetate copolymer) pellets in each cornea.

Other methods for testing PEGylated murine interferon-beta foranti-angiogenic effects in an animal model include (but are not limitedto) protocols for screening new potential anticancer agents as describedin the original Cancer Chemotherapy Reports, Part 3, Vol. 3, No. 2,September 1972 and the supplement In Vivo Cancer Models, 1976-1982, NIHPublication No. 84-2635, February 1984.

Because of the species barriers of Type I interferons, to assess theanti-angiogenic activity of polymer conjugated interferon-beta in rodentmodels, polymer conjugated rodent interferon-beta preparations aregenerated. Such screeing methods are exemplified by a protocol to testfor the anti-angiogenic effects of pegylated murine interferon-beta onsubcutaneously-implanted Lewis Lung Carcinoma.

Origin of Tumor Line:

Arose spontaeously in 1951 as a carcinoma of the lung in a C57BL/6mouse.

Summary of Test Procedures: A tumor fragment is implanted subcutaneouslyin the axillary region of a B6D2F1 mouse. The test agent (i.e, aPEGylated interferon of the invention) is administered at various doses,subcutaneously (SC) or intraperitoneally (IP) on multiple days followingtumor implantation. The parameter measured is median survival time.Results are expressed as a percentage of control survival time.

Animals:

-   -   Propagation: C57BL/6 mice.    -   Testing: B6D2F1 mice.    -   Weight: Mice should be within a 3 gm weight range with a minimum        weight of 18 gm for males and 17 gm for females.    -   Sex: One sex is used for all test and control animals in one        experiment.    -   Source: One source, if feasible, for all animals in one        experiment.

Experiment Size:

-   -   Ten animals per test group.

Tumor Transfer:

PROPAGATION:

-   -   Fragment: Prepare a 2-4 mm fragment of a s.c. donor tumor    -   Time: Day 13-15    -   Site: Implant the fragment s.c. in the axillary region with a        puncture in the inguinal region.

TESTING:

-   -   Fragment: Prepare a 2-4 mm fragment of s.c. donor tumor.    -   Time: Day 13-15.    -   Site: Implant the fragment s.c. in the axillary region with a        puncture in the inguinal region.

Testing Schedule:

-   -   Day 0: Implant tumor. Run bacterial cultures. Test positive        control compound in every odd-numbered experiment. Prepare        materials. Record deaths daily.    -   Day 1: Check cultures. Discard experiment if contaminated.        Randomize animals. Treat as instructed (on day 1 and on        following days).    -   Day 2: Recheck cultures. Discard experiment if contaminated.    -   Day 5: Weigh Day 2 and day of initial test agent toxicity        evaluation.    -   Day 14: Control early-death day.    -   Day 48: Control no-take day.    -   Day 60: End and evaluate experiment. Examine lungs grossly for        tumor.

Quality Control:

Schedule the positive control compound (NSC26271 (Cytoxan at a dose of100 mg/kg/injection)) in every odd-numbered experiment, the regimen forwhich is intraperitoneal on Day 1 only. The lower Test/Control limit forthe positive control is 140%. The acceptable untreated control mediansurvival time is 19-35.6 days.

Evaluation:

The parameter measured is median survival time Compute mean animal bodyweights for Day 1 and Day 5, compute Test/Control ratio for all testgroups with. The mean animal body weights for staging day and finalevaluation day are computed. The Test/Control ratio is computed for alltest groups with >65% survivors on Day 5. A Test/Control ratio value<86% indicates toxicity. An excessive body weight change difference(test minus control) may also be used in evaluating toxicity.

Criteria for Activity:

An initial Test/Control ratio greater than or equal to 140% isconsidered necessary to demonstrate moderate activity. A reproducibleTest/Control ratio value of greater than or equal to 150% is consideredsignificant activity.

1. A composition comprising the glycosylated interferon-beta-1a of SEQID NO: 25 coupled to a non-naturally-occurring polymer at an N-terminalend of said glycosylated interferon-beta-1a, said polymer comprising apolyalkylene glycol moiety.
 2. The composition of claim 1, wherein thepolyalkylene moiety is coupled to the interferon-beta by way of a groupselected from an aldehyde group, a maleimide group, a vinylsulfonegroup, a haloacetate group, plurality of histidine residues, a hydrazinegroup and an aminothiol group.
 3. The composition of claim 1, whereinthe interferon-beta-1a of SEQ ID NO: 25 is an inlerferon-beta-1a fusionprotein.
 4. The composition of claim 3, wherein the interferon-beta-1afusion protein comprises a portion of an immunoglobulin molecule.
 5. Acomposition comprising the glycosylated interferon-beta-1a of SEQ ED NO:26 coupled to a non-naturally-occurring polymer at the N-terminus ofsaid glycosylated interferon-beta-1a, said polymer comprising apolyalkylene glycol moiety.
 6. The composition of claim 5, wherein theglycosylated interferon-beta-1a of SEQ ID NO: 26is an interferon-beta-1afusion protein.
 7. The composition of claim 6, wherein theinterferon-beta-1a fusion protein comprises a portion of animmunoglobulin molecule.
 8. A physiologically active interferon-betacomposition comprising a physiologically active interferon-beta-1acomprising the amino acid sequence of SEQ ID NO: 25 coupled to a polymercomprising a polyalkylene glycol moiety, wherein the interferon-beta-1ais coupled to the polymer at a site on the interferon-beta-1a that is anN- terminal end, wherein the physiologically active interferon-beta 1aand the polyalkylene glycol moiety are arranged such that thephysiologically active interferon-beta-1a in the physiologically activeinterferon-beta composition has an activity at least 2-fold greaterrelative to physiologically active interferon-beta-1b, when measured byan antiviral assay.
 9. The composition of claim 8, wherein theinterferon-beta-1a is coupled to the polymer at a site by way of aglycan moiety the interferon-beta-1a.
 10. The composition of claim 8,wherein interferon-beta-1a is an interferon-beta-1a fusion protein. 11.The composition of claim 10, wherein the interferon-beta-1a fusionprotein comprises a portion of an immunoglobulin molecule.
 12. Aphysiologically active interferon-beta composition comprising apysiologically active glycosylated interferon-beta-1a comprising theamino acid sequence of SEQ ID NO: 25 N-terminally coupled to a polymercomprising a polyalkylene glycol moiety. wherein the physiologicallyactive interferon-beta-1a and the polyalkylene glycol moiety arearranged such that the physiologically active interferon-beta-1a in thephysiologically active interferon-beta composition has equal activityrelative to physiologically active interferon-beta lacking said moiety,when measured by an antiviral assay.
 13. The composition of claim 12,wherein the interferon-beta is coupled to the polymer at a site by wayof a glycan moiety on the interferon-beta.
 14. The composition of claim12, wherein the interferon-beta-1a is an interferon beta fusion protein.15. The composition of claim 14, wherein the interferon beta fusionprotein comprises a portion of an immunoglobulin molecule.
 16. A stable,aqueously soluble, conjugated interferon-beta-1a complex comprising ainterferon-beta-1a comprising the amino acid sequence of SEQ ID NO: 25N-terminally coupled to a polyethylene glycol moiety, wherein theinterferon-beta-1a is coupled to the polyethylene glycol moiety by alabile bond, wherein the labile bond is cleavable by biochemicalhydrolysis and/or proteolysis.
 17. A interferon-beta compositionaccording to claims 12, or 16, wherein the polymer has a molecularweight of from about 5 to about 40 kilodaltons.
 18. A pharmaceuticalcomposition comprising the interferon-beta composition of claim
 17. 19.A physiologically active interferon-beta composition comprising aphysiologically active interferon-beta-1a comprising the amino acidsequence of SEQ ID NO:26 coupled to a non-naturally-occurring polymer atthe N-terminus of said glycosylated interferon-beta-1a, said polymercomprising a polyalkylene glycol moiety wherein the physiologicallyactive interferon-beta-1a and the polyalkylene glycol moiety arearranged such that the physiologically active interferon-beta-1a in thephysiologically active interferon-beta composition has an activity atleast 2-fold greater relative to physiologically activeinterferon-beta-1b, when measured by an antiviral assay.
 20. Thecomposition of claim 19, wherein the interferon-beta-1a is aninterferon-beta-1a fusion protein.
 21. The composition of claim 20,wherein the interferon-beta-1a fusion protein comprises a portion of animmunoglobulin molecule.
 22. A physiologically active interferon-betacomposition comprising a physiologically active glycosylatedinterferon-beta-1a, comprising the amino acid sequence of SEQ ID NO: 25,N-terminally coupled to a polymer comprising a polyalkylene glycolmoiety, wherein the physiologically active interferon-beta-1a and thepolyalkylene glycol moiety are arranged such that the physiologicallyactive interferon-beta-1a in the physiologically active interferon-betacomposition has equal activity relative to physiologicaily activeinterferon-beta lacking said moiety, when measured by an antiviralassay.
 23. The composition of claim 22, wherein the interferon-beta-1ais an interferon beta fusion protein.
 24. The composition of claim 23,wherein the interferon beta fusion protein comprises a portion of animmunoglobulin molecule.